CN1235609A - 免疫刺激性核酸分子 - Google Patents

免疫刺激性核酸分子 Download PDF

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CN1235609A
CN1235609A CN97199352A CN97199352A CN1235609A CN 1235609 A CN1235609 A CN 1235609A CN 97199352 A CN97199352 A CN 97199352A CN 97199352 A CN97199352 A CN 97199352A CN 1235609 A CN1235609 A CN 1235609A
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odn
dna
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阿撒尔·M·科瑞格
约尔·N·科里尼
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Abstract

本发明涉及含有未甲基化的CpG二核苷酸的核酸序列,其中的CpG二核苷酸调节免疫反应,包括刺激Th1类型的免疫活化、细胞因子的产生、NK溶胞活性、以及B细胞的繁殖。这些序列可以用作合成附剂。

Description

免疫刺激性核酸分子
本发明的研究部分地得到了美国国家卫生研究院的第R29-AR42556-01拨款的资助。美国政府有本发明的部分权利。本发明的技术领域
本发明一般地涉及寡核苷酸，具体地讲，涉及那些包括至少一个免疫刺激性的未甲基化CpG二核苷酸的寡核苷酸序列。本发明的背景技术
在二十世纪七十年代，一些研究人员报道了高分子量DNA与细胞膜的结合(Lerner,R.A.et al.,1971.“Membran-associatedDNA in the cytoplasm of diploid human lymphocytes”.Proc.Natl.Acad.Sci.USA68:l12；Agrawal,S.K.,R.W.Wagner,P.K.McAllister,and B.Rosenberg.1975.“Cell-surface-associatednucleic acid in tumorigenic cells made visible with platinum-pyrimidine complexes by electron microscopy”.Proc.Natl.Acad.Sci.USA72:928)。在1985年，Bennett等人首次提出证据证明了DNA与淋巴细胞的结合相似于配体与受体的相互作用，即结合是饱和的、竞争性的、并导致DNA细胞内吞作用和降解为寡核苷酸(Bennett,R.M.,G.T.Gabor,and M.M.Merritt.1985.“DNA binding to human leukocytes.Evidence for receptor-mediated association,internalization,and degradation of DNA”.J.Clin.Invest.76:2182)。类似于DNA，寡脱氧核糖核酸(ODN)也能以饱和、不依赖序列、但依赖温度和能量的形式进入到细胞中(见Jaroszewski,J.W.,and J.S.Cohen.1991.“Cellular uptakeof antisense oligodeoxynucleotides”.Advanced Drug DeliveryReviews6:235；Akhtar,S.,Y.Shoji,and R.L.Juliano.1992.“Pharmaceutical aspects of the biological stability and membranetransport characteristics of antisense oligonucleotides”.In:GeneRegulation:Biology of Antisense RNA and DNA.R.P.Erickson,and J.G.Izant,eds.Raven Press,Ltd.New York,pp.133:and Zhao,Ql,T.Waldschmidt,E.Fisher,C.J.Herrera,and A.M.Krieg.,1994.“Stage specific oligonucleotide uptake in murine bone marrow Bcell precursors”.Blood,84:3660)。但是，还没有克隆化到吸收DNA或ODN的受体，而且，还不清楚ODN结合及细胞吸收是采取与高分子量的DNA相同或不同的机制。
淋巴细胞的ODN吸收已经被证明可由细胞活化来调节。用B细胞分裂素LPS刺激的脾细胞在B细胞群体中显示了ODN吸收的显著提高，而用T细胞分裂素Con A处理的脾细胞显示的ODN吸收的提高是在T细胞中而不是在B细胞中(Krieg,A.M.,F.Gmelig-Meyling,M.F.Gourley,W.J.Kisch,L.A.Chrisey,and A.D.Steinberg.1991.“Uptake of oligodeoxyribonucleotidesby lymphoid cells is heterogeneous and inducible”.AntisenseResearch and Development 1:161)。
已经对一些多聚核苷酸作为反应修饰剂进行了广泛的研究。也许最好的例子是多聚(I,C)，这是一种IFN生产的强力诱导子，也是巨嗜细胞活化子和NK活性诱导子(Talmadge,J.E.,J.Adams,H.Phillips,M.Collins,B.Lenz,M.Schneider,E.Schlick,R.Ruffmann,R.H.Wiltrout,and M.A.Chirigos.1985.“Immunomodulatory effects in mice of polyinosinic-polycytidylicacid comlexed with poly-L-lysine and carboxymethylcellulose”.Cancer Res.“Immunomodulation of natural killer activity bypolyribonucleotides”.J.Biol.Resp.Mod.4:512；Krown,S.E.1986.“Interferons and interferon inducers in cancer treatment”.Sem.Oncol.13:207；and Ewel,C.H.,S.J.Urba,W.C.Kopp,J.W.SmithII,R.G.Steis,J.L.Rossio,D.L.Longo,M.J.Jones,W.G.Alvord,C.M.Pinsky,J.M.Beveridge,K.L.McNitt,and S.P.Creekmore.1992.“Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin-2in patients with cancer:clinical and immunological effects”.Canc.Res.52:3005)。看起来，这种鼠NK活化的单一原因是引入了IFN-β分泌(Ishikawa,R.,and C.A.Biron.1993.“IFN inductionand associated changes in splenic leukocyte distribution”.J.Immunol.150:3713)。这种活化是对核糖为特异的，因为对脱氧核糖没有效力。其体外抗肿瘤活性的效力导致多种采用多聚(I,C)复合多聚-L-赖氨酸和羧甲基纤维素(用以减少RNAse降解)的临床研究(Talmadge,J.E.,et al.,1985.出处同上；Wiltrout,R.H.,et al.,1985.出处同上)；Krown,S.E.,1986.出处同上)；and Ewel.C.H.,et al.,1992.出处同上)。不幸的是，对毒性副作用的研究完全排除了多聚(I,C)成为有用的治疗药剂的可能。
在C8位发生溴或硫醇基取代的鸟嘌呤核糖核苷酸是B细胞的分裂素，并且可以取代“B细胞分化因子”(Feidbush,T.L.,andZ.K.Ballas.1985.“Lymphokine-like activity of8-mercaptoguanosine:induction of T and B cell differentiation”.J.Immunol.134:3204；and Goodman, M.G.1986.“Mechnism ofsnyergy between T cell signals and C8-substituted guaninenucleosides in humoral immunity:B lymphotropic cytokines induceresponsiveness to 8-mercaptoguanosine”.J.Immunol.136:3335 )。8-巯基鸟苷和8-溴鸟苷还可以取代细胞因子来用在MHC限制的CTL的产生(Feldbush,T.L.,1985.出处同上)，增大鼠NK活性(Koo,G.C.,M.E.Jewell,C.L.Manyak,N.H.Sigal,and L.S.Wicker.1988.“Activation of murine natural killer cells andmacrophages by8-bromoguanosine”.J.Immunol.140:3249)，以及与IL-2一起在诱发鼠LAK产生中的协同效应(Thompson,R.A.,and Z.K.Ballas.1990.“Lymphokine-activated killer(LAK)cells.V.8-Mercaptoguanosine as an IL-2-sparing agent inLAK generation”.J.Immunol.145:3524)。这些C8取代的鸟苷的NK和LAK活性的增大看起来归因于它们对IFN的引入(Thompson,R.A.,et al.,1990.出处同上)。近来，由分枝杆菌产生的5’三磷酸化胸苷被发现是对人γδT细胞亚类为促有丝分裂的(Constant,P.,F.Davodeau,M.A.Peyrat,Y.Poquet,G.Puzo,M.Bonneville,and J.-J.Fournie.1994.“Stimulation of humanγδTcells by nonpeptidic mycobacterial ligands”Science264:267)。该篇报道指明，免疫系统可能产生对微生物核酸的带有倾向性的反应方式。
一些研究已经表明，某些DNA结构可以还带有活化淋巴细胞的潜力。例如，Bell等人曾经报道，在脾细胞上清液中的核小体蛋白质-DNA复合物(不是裸露的DNA)引起了B细胞的繁殖和免疫球蛋白的分泌(Bell,DA.,B.Morrison,and P.VanderBygaart.1990.“Immunogenic DNA-related factors”.J.Clin.Invest.85:1487)。在其它的情况下，裸露的DNA已经被报道具有免疫效应。例如，Messina等人最近曾报道说，多聚(dG)·(dC)和多聚(dG·dC)的260至800bp的片段是对B细胞为促有丝分裂的(Messina,J.P.,G.S.Gilkeson,and D.S.Pisetsky.1993.“Theinfluence of DNA structure on the in vitro stimulation of murinelymphocytes by natural and synthetic polynucleotide antigens”.Cell.Immunol.147:148)。Tokunaga等人已经报道了dG·dC诱导γ-IFN和NK活性(Tokunaga,S.Yamamoto,and K.Namba.1988.“A synthetic single-stranded DNA,poly(dG,dC),inducesinterferon-α/b and-g,augments natural killer activity,andsuppresses tumor growth”Jpn.J.Cancer Res.79:682)。除了这些人造的均聚体序列之外，Pisetsky等人报道了纯净哺乳动物DNA没有可检测性免疫效应，但是，从某些细菌来的DNA则诱导了B细胞的活化和免疫球蛋白的分泌(Messina,J.P.,G.S.Gilkeson,and D.S.Pisetsky.1991.“Stimulation of in vitro murinelymphocyte proliferation by bacterial DNA”.J.Immunol.147:1759)。假设这些数据不是那些特异的污染物的结果，那么，这些研究则建议，细菌DNA的特定结构或它们的特定性质使其能够启动B细胞的活化。对分枝杆菌DNA序列的研究显示，含有某种回文序列的ODN可以活化NK细胞(Yamamoto,S.,T.Yamamoto,T.Kataoka,E.Kuramoto,O.Yano,and T.Tokunaga.1992.“Unique palindromic sequences in syntheticoligonucleotides are required to induce INF and agument INF-meidated natural killer activity”.J.Immunol.148:4072；Kuramoto,E.,O.Yano,Y.Kimura,M.Baba,T.Makino,S.Yamamoto,T.Yamamoto,T.Kataoka,and T.Tokunaga.1991.“Oligonucleotidesequences required fro natural killer cell activation”.Jpn.J.CancerRes.83:1128)。
还报道了其它一些硫代磷酸酯修饰的ODN在体外或体内对B细胞的刺激(Tanaka,T.,C.C.Chu,and W.E.Paul.1992.“Anantisense oligonucleotide complementary to a sequence in Ig2bincreases g2b germline transcripts,stimulates B cell DNA synthesis,and inhibits immunoglobulin secretion”.J.Exp.Med.175:597；Branda,R.F.,A.L.Moore,L.Mathews,J.J.McCormack,and G.Zon.1993.“Immune stimulaiton by an antisense oligomercomplementary to the rev gene of HIV-1”.Biochem.Pharmacol.45:2037；McIntyre,K.W.,K.Lombard-Gillooly,J.R.Perez,C.Kunsch,U.M.Sarmiento,J.D.Larigan,K.T.Landreth,and R.Narayanan.1993.“A sense phosphorothioate oligonucleotidedirected to the initiation codon of transcription factor NF-BT65causes sequence-specific immune stimulation”.Antisense Res.Develop.3:309；and Pisetsky,D.S.,and C.F.Reich.1993.“Stimulation of murine lymphocyte proliferation by aphosphorothioate oligonucleotide with antisense activity for herpessimplex virus”.Life Sciences54:101)。这些报道没有建议出在这些ODN中有可以解释这些效果的共同的结构基元或序列因素。
cAMP效应元件结合蛋白质(CREB)和活化转录因子(ATF)或转录因子的CREB/ATF家族是普遍被表达的一类转录因子，其11个成员已经被克隆化了(reviewed in de Groot,R.P.,and P.Sassone-Corsi:“Hormonal control of gene expression:Multiplicity and versatility of cyclic adenosine 3’,5’-monophosphate-responsive nucler regulators”.Mol.Endocrin.7:145,1993；Lee,K.A.W.,and N.Masson:“Transcriptionalregulation by CREB and its relatives”.Biochim.Biophys.Acta1174:221,1993)。它们都属于蛋白质中碱性区/亮氨酸拉链结构(bZip)类。所有的细胞都显示出对一种或一种以上的CREB/ATF蛋白质的表达，但是，所表达的成员和对mRNA剪接的调节则显示为组织特异性的。对活化区的分化剪接可以确定某一具体的CREB/ATF蛋白质是转录抑制子或活化子。许多的CREB/ATF蛋白质活化病毒性转录，但是，有些缺少活化区的剪接变体则是抑制性的。CREB/ATF蛋白质可以作为均二聚体或异二聚体通过cAMP效应元件来结合DNA，而cAMP效应元件，即CRE，其共有形式是未甲基化序列TGACGTC(如果CpG是甲基化的，则结合就被抑制)(Iguchi-Ariga,S.M.M.,andW.Schaffner:″CpG methylation of the cAMP responsiveenhancer/promoter sequence TGACGTCA abolishes specificefactor binding as well as transcriptional activation″.Genes &Develop.3:612,1989.)。
CRE的转录活性在B细胞活化中被提高(Xie,H.T.C.Chiles,and T.L.Rothstein:″Induction of CREB activity via the surface Igreceptor of B cells″.J.Immunol.151:880,1993)。CREB/ATF蛋白质好象是通过CRE来对多种基因的表达进行调节，包括免疫学上重要的基因，例如，fos、jun B、Rb-1、IL-6、IL-1(Tsukada,J.,K.Saito,W.R.Waterman,A.C.Webb,and P.E.Auron:″Transcription factors NF-IL-6 and CREB recognize a commonessential site in the human prointerleukin 1 gene″.Mol.Cell.Biol.14:7285,1994；Gray,G.D.,O.M.Hernadez,D.Hebel.M.Root,J.M.Pow-Sang,and E.Wickstrom:″antisense DNA inhibition oftumor growth induced by c-Ha-ras oncogene in nude mice″.CancerRes.53:577,1993),IFN-(Du,W.,and T.Maniatis:″AnTF/CREB binding site protein is required for virus induction ofhuman interferon B gene″.Proc.Natl.Acad.Sci.USA 89:2150,1992),TGF-1(Asiedu,C.K.,L.Scott,R.K.Assoian,M.Ehrlich:＂Binding of AP-1/CREB proteins and of MDBP to contiguous sitesdownstream of the human TGF-B1 gene″.Biochim.Biophys.Acta1219:55,1994),TGF-2,Ⅱ MHC类(Cox,P.M.,and C.R.Goding:″An ATF/CREB binding motif is required for aberrant constitutiveexpression of the MHC class Ⅱ DRa promoter and activation bySV40T-antigeh″.Nucl.Acids Res.20:4881,1992)，E-选择素，GM-CSF,CD-8，种系Ig恒定区基因，TCR V基因，以及繁殖细胞核抗原(Huang,D.,P.M.Shipman-Appasamy,D.J.Orten,S.H.Hinrichs,and M.B.Prystowsky:″Promoter activity of theproliferating-cell nuclear antigen gene is associated with inducibleCRE-binding proteins in interleukin 2-stimulated T lymphocytes:.Mol.Cell.Biol.14:4233,1994)。除了通过cAMP途径的活化，CREB还可以介导对细胞内Ca++浓度变化的转录应答(Sheng,M.,G.McFadden,and M.E.Greenberg:″Membrane depolarizationand calcium induce c-fos transeription via phosphorylation oftranseription fector CREB″.Neuron4:571,1990)。
在CREB/ATF蛋白质的转录活化中，蛋白质-蛋白质之间的相互作用显示为极其重要角色。已经有一些发表的研究报道了在NFKB蛋白质和CREB/ATF蛋白质之间的直接性或间接性相互作用[Whitley,et al.,(1994)Mol.& Cell.Biol.14:6464；Cogswell,et al.,(1994)J.Immun.153:712；Hines,et al.,(1993)Oncogene8:3189；and Du,et al.,(1993)Cell74:887]。通过环AMP途径对CREB的活化需要蛋白质激酶A(PKA)，其将CREB341在ser133磷酸化，并允许其与近来克隆化的蛋白质CBP结合(Kwok,R.P.S.,J.R.Lundblad,J.C.Chrivia,J.P.Richards,H.P.Bachinger,R.G.Brennan,S.G.E.Roberts,M.R.Green,and R.H.Goodman:″Nuclear protein CBP is a coactivator for thetranscription factor CREB″.Nature370:223,1994；Arias,J.,A.S.Alberrs,P.Brindle,F.X.Claret,T.Smea,M.Karin,J.Feranmisco,and M.Montminy:″Activation of cAMP and mitogen responsivegenes relies on a common nuclear factor″.Nature370:226,1994.)。CBP随后再与碱性转录因子TFIIB相互作用造成提高了的转录。CREB还被报道与dTAFII110相互作用，该dTAFⅡ110是一种TATA结合蛋白质相关的因子，其结合可以调节转录(Ferreri,K.,G.Gill,and M.Montminy:″The cAMP-regulatedtranscription factor CREB interacts with a component of the TFⅡDcomplex″.Proc.Natl.Acad.Sci.USA91:1210,1994)。除了这些相互作用之外，CREB/ATF蛋白质可以特异性结合多种其它的核因子(Hoeffler,J.P.,J.W.Lustbader,and C.-Y.Chen:″Identification of multiple nuclear factors that interact with cyclicadenosine 3′,5′-monohosphate response element-binding proteinand activating transcription factor-2 by protein-proteininteractions″.Mol.Endocrinol.5:256,1991)，但是，这些相互作用中的大多数的生物学重要性还都是未知的。一般都认为CREB与DNA的结合是均二聚体的或与其它的蛋白质的异二聚体。另人惊奇的是，CREB单体对转录的活化是组成性的(Krajewski,Wl,and K.A.W.Lee:″A monomeric derivative of the cellulartranscription factor CREB functions as constitutive activator″.Mol.Cell.Biol.14:7204,1994)。
除了在调节细胞转录中的重要作用之外，最近还证实了CREB/ATF蛋白质被某些感染性病毒和逆病毒所破坏，使其需要病毒复制。例如，在已知的最为强烈的哺乳动物启动子中，即巨细胞病毒立即早期启动子中，含有对启动子功能为必须的CRE的11个拷贝(Chang,Y.-N.,S.Crawfbrd,J.Stall,D.R.Rawlins,K.-T.Jeang,and G.S.Hayward:″The palindromic series Irepeats in the simian cytomegalovirus major immediate-earlypromoter behave as both strong basal enhancers and cyclic AMPresponse elements″.J.Virol.64:264,1990)。诱导众多启动子的腺病毒ElA蛋白质的至少一部分转录活化效应归因于其与CREB/ATF蛋白质的DNA结合区的结合，即与ATF-2的结合，其介导ElA可诱导的转录活化(Liu,F.,and M.R.Green:″Promoter targeting by adenovirus Ela through interaction withdifferent cellular DNA-binding domains″.Nature368:520,1994)。还建议的是，ElA与CREB结合蛋白质CBP相结合(Arany,Z.,W.R.Sellers,D.M.Livingston,and R.Eckner:″ElA-associated p300and CREB-associated CBP belong to a conservedfamily of coactivators″.Cell77:799,1994)。人T嗜淋巴细胞病毒-I(HTLV-1)，即造成人T细胞白血病和热带痉挛轻瘫的逆病毒，也需要CREB/ATF蛋白质来复制。在这种情况下，逆病毒产生蛋白质Tax，其与CREB/ATF蛋白质结合，再指引它们从其正常的细胞结合位点改变为存在于HTLV转录增强子中的不同的DNA序列(侧翼序列为G-和C-富集的序列)(Paca-Uccaralertkun,S.,L.-J.Zhao,N.Adya,J.V.Cross,B.R.Cullen,I.M.Boros.and C.-Z.Giam:″In vitro selection of DNA elementshighly responsive to the human T-cell lymphotropic virus type Itranscriptional activator, Tax″. Mol.Cell.Biol.14:456,1994；Adya,N.,L.-J.Zhao,W.Huang,I.Boros,and C.-Z.Giam:″Expansion ofCREB′s DNA recognition specificity by Tax results frominteraction with Ala-Ala-Arg at positions 282-284 near theconserved DNA-binding domain of CREB″.Proc.Natl.Acad.Sci.USA91:5642,1994) 。本发明的概述
本发明的基础是发现了某些含有未甲基化的胞嘧啶-鸟苷(CpG)二核苷酸活化对象中的淋巴细胞并使对象的免疫系统由Th2转变为Th1(例如，诱导单核细胞以及其它细胞产生Th1细胞因子，包括IL-12、IFN-γ、和GM-CSF)。基于这个发现，本发明在一方面涉及新的免疫刺激性核酸组合物。
在一个实施方案中，本发明提供了被分离的免疫刺激性的含有CpG基元的核酸序列，其表达式为：
                 5′N1X1CGX2N23′其中，至少有一个核苷酸将连续的CpGs间隔开来；X1是腺嘌呤，鸟嘌呤，或胸腺嘧啶；X2是胞嘧啶或胸腺嘧啶；N是任何的核苷酸，且N1+N2是0-26碱基，条件是N1和N2都不含有CCGG四元体或多于一个的CCG或CGG三元体；并且核酸序列的长度为约8-30碱基对。
在另一个实施方案中，本发明提供了被分离的免疫刺激性的含有CpG基元的核酸序列，其表达式为：
             5′N1X1X2CGX3X4N23′其中，至少有一个核苷酸将连续的CpGs间隔开来；X1X2是选自GpT、GpG、GpA、ApT和ApA；X3X4是选自TpT或CpT；N是任何的核苷酸，且N1+N2是0-26碱基，条件是N1和N2都不含有CCGG四元体或多于一个的CCG或CGG三元体；并且核酸序列的长度为约8-30碱基对。
在另一个实施方案中，本发明提供了刺激免疫活化的方法，将本发明的核酸序列提供给对象，优选人类。在优选的实施方案中，免疫活化产生主要是Th1形式的免疫活化。
在另一个实施方案中，本发明的核酸序列刺激细胞因子的生产。具体地讲，IL-6、IL-12、IFN-γ、TNF-α和GM-CSF是通过用本文中所述的核酸序列刺激免疫系统来产生的。在另一方面，本发明的核酸序列刺激天然杀死细胞(NK)的溶胞活性和B细胞的繁殖。
在另一个实施方案中，本发明的核酸序列被用作人造附剂来在哺乳动物如小鼠或人中产生抗体。
在另一个实施方案中，通过抑制对象的对CpG介导的淋巴细胞活化的应答来治疗自体免疫紊乱。本发明提供了施加对体内酸化的抑制物如bafilomycina，氯喹，和莫能菌素来缓解自体免疫紊乱。具体地讲，用这种方式治疗了系统性红斑狼苍。
本发明的核酸序列还可以用于治疗、防止或缓解其它的紊乱(例如，肿瘤或癌症，病毒性、真菌性、细菌性或寄生性感染)。此外，本发明的核酸序列可以施加给对象来刺激其对疫苗的应答。进一步，将对象的免疫系统从Th2改变为Th1，本发明的核酸序列可以治疗和防止气喘疾病。此外，本发明的核酸序列可以施加给患有过敏疾病的对象，作为消除敏感的制剂来治疗或防止与气喘相关的过敏反应。
进一步，本发明的核酸序列的诱导白血细胞进入细胞循环的能力支持了其治疗白血病的应用，即提高了在常规的消除化疗之后的白血细胞的敏感性，或者将本发明的核酸序列与其它的免疫治疗物一起使用。
本发明的其它的特点和优点将可以从下面的描述和权利要求书中更为明显地看出。附图的简要说明
图1A-C用制图说明在消除了T细胞的脾细胞培养物中对各种DNA序列的应答的IL-6生产的剂量依赖情况。
图1A是大肠杆菌DNA(1)和小牛胸腺DNA(n)序列和LPS(10倍浓度的大肠杆菌和小牛胸腺DNA)(u)。
图1B是对照的磷酸二脂寡聚脱氧核苷酸(ODN)5′ATGGAAGGTCCAGTGTTCTC3′(SEQ ID No:1)(n)和两个磷酸二脂CpG ODN 5′ATCGACCTACGTGCGTTCTC3′(SEQ IDNo:2)(u)和5′TCCATAACGTTCCTGATGCT3′(SEQ ID No:3)(1)。
图1C是对照的硫代磷酸酯ODN 5′GCTAGATGTTAGCGT3′(SEQ ID No:4)(n)和两个硫代磷酸酯CpG ODN5′GAGAACGTCGACCTTCGAT3′(SEQ ID No:5)(u)和5′GCATGACGTTGAGCT3′(SEQ ID No:6)(1)。数据代表三份的平均值±标准偏差。
图2图示注射后1-8小时所确定的体内CpG DNA诱导的IL-6生产。数据代表从两个小鼠的血清的两次分析的平均值。对BALB/c小鼠(每组两只)静脉内注射100微升的PBS(o)或200微克的CpG硫代磷酸酯0DN5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)(n)或非-CpG硫代磷酸酯ODN5′TCCATGAGCTTCCTGAGTCT3′(SEQ ID No:8)(u)。
图3是放射性自显影图，说明IL-6mRNA表达，由反转录聚合酶链反应确定，在肝、脾和胸腺中各个时间的情况，事先对BALB/c小鼠(每组两只)进行体内刺激，静脉内注射100微升的PBS,200微克的CpG硫代磷酸酯ODN5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)非-CpG硫代磷酸酯ODN5′TCCATGAGCTTCCTGAGTCT3′(SEQ ID No:8)。
图4A是图示抗IL-6对CpG诱导的IgM生产的剂量依赖性抑制。从DBA/2小鼠来的脾B细胞用CpG ODN5′TCCAAGACGTTCCTGATGCT3′(SEQ ID No:9)刺激，有给出浓度的中性抗-IL-6(u)或等模标本对照Ab(1)的存在，在培养物上清液中的IgM水平有ELISA确定。在没有CpG ODN存在的情况下，抗-IL-6抗体对IgM分泌没有效应(n)。
图4B图示用CpG S-ODN5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)和抗-IL-6(u)培养或用抗-IL-6抗体单独培养(n)的脾B细胞CpG-诱导的刺激指数。数据代表三份的平局值±标准偏差。
图5是条块图，说明在wEHI-231细胞中的氯霉素酰基转移酶(CAT)的活性，其中的细胞被不含启动子的CAT结构(pCAT)、阳性对照质粒(RSV)、IL-6启动子-CAT结构自身、以及按照给定浓度的CpG5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)或非-CpG5′TCCATGAGCTTCCTGAGTCT3′(SEQ ID No:8)硫代磷酸酯ODN与IL-6启动子-CAT结构所转染。数据代表三份数据的平均值。
图6是含有免疫刺激性的未甲基化CpG的核酸的免疫效果的总体图示，该核酸可以直接活化B细胞和单核细胞(包括巨嗜细胞和树状细胞)。免疫刺激性寡核苷酸不直接活化纯化的NK细胞，但可以使它们适合于应答IL-12并使IFN-γ生产有明显提高。由于使NK细胞被诱导产生IL-12和随后的IFN-γ分泌被提高，免疫刺激性核酸促进了Th1类型的免疫应答。没有发现高纯度的T细胞可以直接活化细胞因子分泌的增加。然而，由免疫刺激性寡核苷酸所诱导的Th1细胞因子分泌促进了细胞毒性淋巴细胞应答的产生。
图7是自显影照相图，显示NFkB mRNA在单核细胞中的诱导结果，这些细胞被大肠杆菌(EC)DNA(含有未甲基化CpG基元)、对照(CT)DNA(不含未甲基化CpG基元)和脂多糖(LPS)等所处理并分别在接触后的第15分钟、30分钟等各时间测量。
图8A显示流式细胞仪的测定结果，使用的是小鼠B细胞，用二倾若丹明123染色来确定反应性氧的种类和水平。在A组中的仅用了染料的情况显示细胞对染料的阳性背景值为28.6％。这个水平的反应性氧种类在用PMA和离子霉素处理20分钟的细胞中被提高到了80％，即阳性对照(B组)。细胞用CpG寡核苷酸(TCCATGACGTTCCTGACGTT,SEQ ID No.10)处理后也显示了反应性氧种类水平的提高，即50％以上的细胞变为阳性(D组)。然而，用同样序列的寡核苷酸处理但CpGs被换为(TCCATGAGCTTCCTGAGTGCT,SEQ ID No.11)则没有显示反应性氧种类水平的提高(E组)。
图8B显示流式细胞仪的结果，使用了小鼠B细胞，有氯喹存在，用二氢若丹明123染色来确定反应性氧种类的水平。氯喹将细胞中背景的反应性氧种类的水平稍微降低了一些，使在A组中的未处理的细胞仅有4.3％为阳性。氯喹完全终止了在用CpG DNA处理的细胞(B组)中对反应性氧种类的诱导，但对用PMA和离子霉素处理的细胞(E组)则没有降低其反应性氧种类水平。
图9说明用肺灌洗细胞计数对时间的作图。该图显示，当小鼠开始被注射了Schistosoma mansoni卵这种诱导Th2应答的“卵”并且随后呼吸Schistosoma mansoni卵抗原“SEA”后(空心环)，则在肺部内有众多的发炎的细胞。然而，当小鼠开始接受的是CpG寡核苷酸(SEQ ID No.10)以及卵时，则肺部内的发炎细胞并不随呼吸SEA而提高(空心三角形)。
图10说明肺部灌洗嗜伊红粒细胞计数对时间的作图。该图显示，当小鼠开始注射了卵并随后呼吸SEA(空心环)，则在肺部内有众多的嗜伊红粒细胞存在。然而，当小鼠开始接受的是CpG寡核苷酸(SEQ ID No.10)和卵时，则在肺部内的发炎细胞就不随着后来呼吸SEA而增高(空心三角形)。
图11是条块图，说明巨嗜细胞、淋巴细胞、嗜碱性细胞、嗜伊红粒细胞分别暴露于盐水、卵加SEA、卵加SEQ ID No.11及随后的SEA、卵加对照寡核苷酸(SEQ ID No.11)以及随后的SEA后，诱导的细胞百分数的效应。当小鼠在暴露于卵的同时给予对照寡核苷酸，则吸入SEA后对嗜伊红粒细胞在肺部内没有明显效应。因此，当小鼠在第14天或第21天呼吸卵，它们在肺部内产生急性发炎。然而，如果在第0天或第7天开始接触抗原的时候将CpG寡核苷酸同卵一起提供给小鼠，则就完全防止小鼠在随后的第14天呼吸卵抗原所产生的嗜伊红粒细胞增加。
图12是一个条块图，说明注射不同剂量的保护性寡核苷酸SEQ ID No.10所产生的嗜伊红粒细胞计数的情况。
图13是随时间的小鼠产生白细胞介素-4(IL-4)的情况(pg/ml)，其所应答的分别是注射卵和随后的SEA(空心菱形)；卵加SEQ ID No.10和随后的SEA(空心环)；盐水加随后的盐水(空心矩形)。该图的结果显示，所产生的发炎的应答相关于在肺部的Th2细胞因子IL-4的水平。
图14是条块图，说明随时间的小鼠产生白细胞介素-12(IL-12)的情况(pg/ml)，其所应答的分别是注射盐水；卵和随后的SEA；卵加SEQ ID No.10和随后的SEA。该图的结果显示，注射含有未甲基化CpG基元的寡核苷酸可以实际上使肺对细胞因子的应答变为产生IL-12，表明是Th1类型的免疫应答。
图15是条块图，说明随时间的小鼠产生伽玛干扰素(IFN-γ)的情况(pg/ml)，其所应答的分别是注射盐水；卵和随后的SEA；卵加SEQ ID No.10和随后的SEA。该图的结果显示，注射含有未甲基化CpG基元的寡核苷酸还可以使肺对细胞因子的应答变为产生IFN-γ，表明是Th1类型的免疫应答。本发明的详细描述定义
在本文中，下面各种术语和词组所表达的意思定义如下：
术语“过敏原”指的是可以在易感染的对象中产生过敏反应或气喘反应的物质。过敏原的名单是非常巨大的，可以包括花粉、昆虫毒物、动物皮、真菌孢子和药物(青霉素)等。天然的、动物的和植物的过敏原的实例包括对下面各属为特异性的蛋白质：犬(Canisf familiaris)，螨(Dermatophagoides farinae),Felis(Felis domesticus)，豚草(Ambrosia artemiisfolia)，黑麦草(Lolium perenne或Lolium multiflorum)，柳杉(Cryptomeriajaponica)，链格孢(Alternaria alternata)，小蠹甲(Alder)，桤木(Alnus gultinosa)，桦木(Betula verrucosa)，栎(Quercusalba)，木犀榄(Olea europa)，蒿(Artemisia vulgaris)，车前(Plantago lanceolata)，墙草(Parietaria officinalis或Parietariajudaica)，小蠊(Blattella germanica)，蜂(APis multiflorum)，柏木(Cupressus sempervirens,Cupressus arizonica和Cupressusmacrocarpa)，刺柏(Juniperus sabinoides,Juniperus virginiana,Juniperus communis和Juniperus ashei),Thuya(Thuyaorientalis)，扁柏(Chamaecyparis obtusa)，大蠊(Periplanetaamericana)，冰草(Agropyron repens)，黑麦(Secale cereale)，小麦(Triticum aestivum)，鸭茅(Dactylis glomerata)，羊茅(Festucaelatior)，早熟禾(Poapratensis或Poa compressa)，燕麦(Avenasativa)，绒毛草(Holcus lanatus)，黄花茅(Anthoxanthumodoratum)，燕麦草(Arrhenatherum elatius)，剪股颖(Agrostisalba)梯牧草(Phleum pratense),(Phalaris arundinacea)，雀稗(Paspalum notatum)，高粱(Sorghum halepensis)，以及雀麦(Bromus inermis)。
术语“过敏”指的是对某种物质(过敏原)所获得的过度敏感。过敏包括湿疹、过敏性鼻炎、枯草热、支气管气喘、寻麻疹、食物过敏，以及其它的特异反应性疾病。
术语“气喘”指的是呼吸道的疾病，其特征是发炎，气管狭窄，对吸入的物质的反应增加。气喘虽然不是必然的但也是经常地伴随着特异反应性或过敏性症状。
术语“免疫系统缺陷”指的是这样的一些疾病或紊乱，其中，个体的免疫系统不能正常发挥功能，或将使该个体的免疫应答不能对消除肿瘤或癌症[例如，脑瘤、肺(包括小细胞和非小细胞)癌、卵巢癌、乳腺癌、前列腺癌、直肠癌、以及其它的恶性瘤和肉瘤]或其它的感染而提高其有用性。
感染性的病毒的实例包括：Retroviridae[例如，人免疫缺陷病毒，如HIV-1(也称为HTLV-Ⅲ,LAV或HTLV-Ⅲ/LAV，或HIV-Ⅲ；以及其它的分离物，例如HIV-LP)]；小RNA病毒科[Picornaviridae(例如，polio病毒，甲肝病毒，肠道病毒，人柯萨奇病毒，鼻病毒)]，嵌杯样病毒[Caliciviridae(例如，造成肠道疾病的品系)]，黄色病毒[Flaviridae，例如，登革病毒(dengue viruses)，脑病毒(encephalitis viruses)，黄热病毒(yellow fever viruses)]，冠形病毒[Coronaviridae，例如冠形病毒(coronaviruses)]，弹状病毒[Rhabdoviridae，例如，vesicular stomatitis viruses,rabies viruses],Filoviridae(例如，ebola viruses)，副粘病毒[Paramyxoviridae，例如，parainfluenzaviruses,mumps virus,measles virus,respiratory syncytialvirus]，亚粘病毒[Orthomyxoviridae，例如，influenza viruses],Bungaviridae[例如，Hantaan viruses,bunga viruses,phleboviruses和Nairo viruses]，嵌沙样病毒[Arena viridae，例如，hemorrhagic fever viruses]，呼吸肠道病毒[Reoviridae，例如，reoviruses,orbiviruses和rotaviruses],Birnaviridae，肝炎病毒[Hepadnaviridae，例如，乙形肝炎病毒(Hepatitis Bvirus)]，细小病毒[Parvoviridae(parvoviruses)]，乳多孔病毒[Papovaviridae，例如，乳突病毒(papilloma viruses,polyomaviruses)]，腺病毒[Adenoviridae(most adenoviruses)]，疱疹病毒[Herpesviridae，例如，herpes simplex virus(HSV)1和2,varicella zoster virus,cytomegalovirus(CMV),herpes viruses]，痘病毒[variola viruses,vaccinia viruses,pox viruses]，和虹色病毒[Iridoviridae，例如，African swine fever virus]，以及一些未分类的病毒[例如，海绵脑病的致病剂，δ肝炎致病剂(被认为是乙型肝炎病毒的缺陷型变体)，非甲非乙肝炎的致病剂(1类=内部传染，2类=肠胃外传染(如丙型肝炎)，Norwalk病毒以及相关的病毒，和星状病毒)]。
感染性细菌的实例包括：Helicobacter pyloris，疏螺旋体(Borelia burgdorferi,Legionella pneumophilia，分杆杆菌[Mycobacteria sps.，例如，结核杆菌(M.Tuberculosis)，鸟型结核分支杆菌(M avium),M.intracellulare,M.kansaii,M.gordonae]，金黄色葡萄球菌(Staphylococcus aureus)，淋病奈瑟氏菌(Neisseria gonorrhoeae)，脑膜炎双球菌(Neisseriameningitidis)，单核细胞增多性李斯特氏菌(Listeriamonocytogenes)，酿脓链球菌[Streptococcus pyogenes(A组Streptococcus)]，无孔链球菌[Streptococcus agalactiae(B组Streptococcus)]，链球菌属[Streptococcs(viridans组)]，粪链球菌(Streptococcus faecalis)，牛链球菌(Streptococcus bovis)，链球菌属(Streptococcus厌氧菌，anaerobic sps.)，肺炎链球菌(Streptococcus pneumoniae)，弯曲杆菌(pathogenicCampylobacter sp.,)，肠球菌(Enterococcus sp.,)，流感嗜血菌(Haemophilus influenzae)，炭疽芽孢杆菌(Bacillusantracis)，白喉棒杆菌(Corynebacterium diphtheriae)，棒杆菌属( Corynebacterium sp.,)，红斑丹毒丝菌(Erysipelothrixrhusiopathiae)，产气英膜梭菌(Clostridium perfringers)，破伤风梭菌(Clostridium tetani),Enterobacter aerogenes，肺炎克雷伯氏菌(Klebsiella pneumoniae)，多杀巴斯德氏菌(Pasturella multocida)，拟杆菌(Bacteroides sp.,)，核粒梭形杆菌(Fusobacterium nucleatum)，念球状链杆菌(Streptobacillus moniliformis)，梅毒螺旋菌(Treponemapallidium)，细弱密螺旋体(Treponema pertenue)，钩端螺旋体(Leptospira)和伊氏放线菌(Actinomyces israelli)。
感染性真菌的实例包括：新型隐球菌(Cryptococcusneoformans)，英膜组织胞浆菌(Histoplasma capsulatum)，粗球孢子菌(Coccidioides immitis)，皮炎芽酵母(Blastomycesdermatitidis)，沙眼衣原体(Chlamydia trachomatis)，白色念球菌(Candida albicans)。其它的感染性有机体(例如，原生动物)包括：恶性疟原虫(Plasmodium falciparum)和鼠弓形虫(Toxoplasma gondii)。
术语“免疫刺激性核酸分子”指的是这样的核酸分子，它们含有未甲基化的胞嘧啶，鸟嘌呤二核苷酸序列(即，“CpGDNA”或含有胞嘧啶并接着鸟苷且由磷酸盐键所连接的DNA)并刺激脊椎动物淋巴细胞(例如，具有促有丝分裂效应，或诱导或提高细胞因子的表达)。免疫刺激性核酸分子可以是双链的或单链的。一般，双链的分子在体内比较稳定，而单链的分子则具有提高了的免疫活性。
在本发明的一个优选的实施方案中，提供了被分离的免疫刺激性核酸序列，其含有的CpG基元由下式表达：
                 5′N1X1CGX2N23′其中，至少有一个核苷酸将连续的CpGs间隔开来；X1是腺嘌呤，鸟嘌呤，或胸腺嘧啶；X2是胞嘧啶或胸腺嘧啶；N是任何的核苷酸，且N1+N2是0-26碱基，条件是N1和N2都不含有CCGG四元体或多于一个的CCG或CGG三元体；并且核酸序列的长度为约8-30碱基对。
在另一个实施方案中，本发明提供了被分离的免疫刺激性的含有CpG基元的核酸序列，其表达式为：
             5′N1X1X2CGX3X4N23′
其中，至少有一个核苷酸将连续的CpGs间隔开来；X1X2是选自GpT、GpG、GpA、ApT和ApA；X3X4是选自TpT或CpT；N是任何的核苷酸，且N1+N2是0-26碱基，条件是N1和N2都不含有CCGG四元体或多于一个的CCG或CGG三元体；并且核酸序列的长度为约8-30碱基对。
优选的是，本发明的免疫刺激性核酸序列包括X1X2，它们选自GpT、GpG、GpA和ApA，且X3X4选自TpT、CpT和GpT(见表5)。为了促进吸收到细胞中，含有免疫刺激性核酸分子的CpG优选为8-30个碱基的长度。然而，如果有足够的免疫刺激性基元的存在，则任何长度的核酸(甚至很多个碱基对长)都可以是免疫刺激性的，因为这样的大型的核酸在细胞内被降解为寡核苷酸。优选的合成性的寡核苷酸不含有CCGG四元体或多于一个的CCG或CGG三元体在其或靠近其5′和/或3′端，和/或其共有的促有丝分裂CpG基元不是回文结构。当寡核苷酸整合有磷酸盐主链的修饰时，用稳定的寡核苷酸可以获得长久的免疫刺激性。例如，当修饰为硫代磷酸酯或硫代磷酸酯类的修饰时既是如此。另外，更具体地讲，磷酸盐主链修饰发生在核酸的5′端，即在核酸5′端的最初两个核苷酸上。进而言之，磷酸盐主链修饰可以发生在核酸的3′端，即在核酸的3′端的最后5个核苷酸上。
优选的免疫刺激性CpG DNA是寡核苷酸时，其长度为8-30个碱基对。另外，CpG二核苷酸可以在质粒中大规模生产，当其被给予个体后，降解为寡核苷酸。优选的免疫刺激性核酸分子[例如，用于提高疫苗的有效性，或在个体内刺激抗体(例如体液的)应答来治疗免疫系统缺陷]具有相对高的对B细胞、单核细胞和/或天然杀死细胞反应的刺激系数(例如，细胞因子、繁殖性、溶胞性或其它的反应)。
本发明的核酸序列刺激对象中的细胞因子的产生。细胞因子包括但不限于IL-6、IL-12、IFN-γ、TNF-α、GM-CSF。这些序列的实例包括：TCCATGTCGCTCCTGATGCT(SEQ ID NO:42),TCCATGTCGTTCCTGATGCT(SEQ ID NO:43)，和TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:56)。
本发明的核酸序列还可以用于刺激天然杀死细胞(NK)在如人的对象中的溶胞活性。具体的但非限制性的实例包括：
TCGTCGTTGTCGTTGTCGTT(SEQ ID NO:57),
TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:58),
TCGTCGTTGTCGTTTTGTCGTT(SEQ ID NO:59),
GCGTGCGTTGTCGTTGTCGTT(SEQ ID NO:),
TGTCGTTTGTCGTTTGTCGTT(SEQ ID NO:),
TGTCGTTGTCGTTGTCGTT(SEQ ID NO:60)和
TCGTCGTCGTCGTT(SEQ ID NO:61)。
本发明的核酸序列还可以用于在如人的对象中刺激B细胞繁殖。具体的但非限制性的实例包括：
TCCTGTCGTTCCTTGTCGTT(SEQ ID NO:62),
TCCTGTCGTTTTTTGTCGTT(SEQ ID NO:63),
TCGTCGCTGTCTGCCCTTCTT(SEQ ID NO:64),
TCGTCGCTGTTGTCGTTTCTT(SEQ ID NO:65),
TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:66),
TCGTCGTTGTCGTTTTGTCGTT(SEQ ID NO:67)，和
TGTCGTTGTCGTTGTCGTT(SEQ ID NO:68)。
在本发明的另一个方面，本发明的核酸序列可以在哺乳动物中生产抗体时作为附剂使用。具体的但非限制性的实例包括TCCATGACGTTCCTGACGTT(SEQ ID NO:10),GTCG(T/C)T和TGTCG(T/C)T。进而言之，这些核酸序列可以用于治疗和防止气喘疾病的症状，将对象的免疫应答从Th2型改变为Th1型。具体的实例包括TCCATGACGTTCCTGACGTT(SEQ IDNO:10)。
具体的免疫刺激性CpG DNA的刺激指数可以用各种免疫细胞检测来测定。优选地，免疫刺激性CpG DNA的刺激指数对B细胞繁殖为约5，优选至少为至少10，更优选地为至少15，最优选地为至少约20，由3H尿苷在B细胞培养物中测定，其中，在37℃下与20μM的ODN接触20小时，并且用1μCi的3H尿苷冲击，并按照实施例1所述在4小时后收获和计数。对于体内的应用，例如，用于治疗免疫系统缺陷刺激细胞介导的对象中的免疫反应，重要的是，免疫刺激性CpG DNA要能够有效地诱导单核细胞分泌细胞因子和/或天然杀死细胞(NK)的细胞溶胞活性。
优选的免疫刺激性CpG核酸应该可以产生至少约500pg/ml的TNF-α、15pg/ml的IFN-γ、70pg/ml的GM-CSF、275pg/ml的IL-6、200pg/ml的IL-12，具体依据治疗指标而定，可以按照实施例2中的所述的检测来确定。其它优选的免疫刺激性CpGDNAs要能够产生至少约10％，更优选至少约15％，而最优选至少约20％的YAC-1细胞特异性溶胞，或至少约30％，更优选至少约35％，和最优选至少约40％的2C11细胞特异性溶胞，由实施例4中描述的检测来确定。
术语“核酸”或“DNA”指的是多核苷酸{即，含有糖(如核糖或脱氧核糖)且通过磷酸盐基团连接到可交换的有机碱上的分子，其替换嘧啶[例如，胞嘧啶(C)、胸腺嘧啶(T)或尿嘧啶(U)]，或替代嘌呤[例如，腺嘌呤(A)，鸟嘌呤(G)]}。在本文中，该术语指的是核糖核苷酸以及寡脱氧核糖核苷酸。该术语应该包括多核苷(即，多核苷酸减去一个磷酸)以及其它的含有有机碱的聚合物。核酸分子可以从已经存在的核酸源获得(例如，基因组或cDNA)，但是，优选合成性的(例如，由寡核苷酸合成所产生的)。
术语“核酸释放复合物”指的是核酸分子(通过离子键或共价键，或包埋方式)与靶向装置[例如，对靶细胞(如B细胞和天然杀死(NK)细胞)表面有较高亲和结合性和/或提高靶细胞的吸收的分子]结合在一起。核酸释放复合物的实例包括与下列物质结合的核酸：固醇(例如，胆固醇)、脂类(例如，阳离子脂、病毒体、或脂质体)、或靶细胞特异性结合制剂(例如，由靶细胞特异性受体所识别的配体)。优选的复合物必须在体内足够地稳定来防止在被靶细胞内化之前就显著地解脱偶联。然而，这些复合物要能够在细胞内适宜的条件下被剪切，使核酸以功能性的形式被释放。
术语“回文序列”指的是反向的重复(即，如ABCDEE′D′C′B′A′这样的序列，其中A和A′是能够形成常规的watson-Crick碱基对的碱基。在体内，这样的序列可以形成双链结构。
术语“稳定化的核酸分子”指的是在体内对降解(例如，通过外切核酸酶或内切核酸酶的降解)具有相对抵抗性的核酸分子。稳定可以是长度的功能或次级结构的功能。长度为几十个甚至上百个碱基的含有未甲基化CpG的核酸分子对体内降解有相对的抵抗性。对于较短的免疫刺激性核酸分子，次级结构可以稳定和提高其功能。例如，如果核酸分子的3′端具有对上游区的自己互补性，则它就可以回折来形成某种茎-环结构，然后，核酸分子变为稳定的并从而表现更强的活性。
优选的本发明的稳定化核酸分子具有被修饰的主链。用于免疫刺激时，特别优选的核酸分子是硫代磷酸酯(即，核酸分子的至少一个磷酸氧被硫所取代)或硫代磷酸酯修饰的核酸分子。更具体地讲，磷酸主链修饰发生在核酸的5′端，在核酸5′端的最初两个核苷酸上。进而言之，磷酸主链修饰可以发生在核酸的3′端，发生在核酸的3′端的最后5个核苷酸上。除了稳定的核酸分子外，正如本文中进一步描述的那样，硫代磷酸酯-修饰(包括二硫代磷酸酯-修饰)的核酸分子可以提高该核酸分子的免疫刺激程度，它们含有所示的未甲基化CpG二核苷酸。题目为“硫代磷酸酯寡核苷酸类似物的免疫刺激”的国际专利申请公开第WO95/26204号也报道了硫代磷酸酯修饰的寡核苷酸的非序列特异性的免疫刺激效果。如本文所描述的，含有未甲基化CpG的并具有硫代磷酸酯主链的核酸分子已经被发现倾向于活化B细胞活性，而含有未甲基化CpG的并具有磷酸二酯主链的核酸分子已经被发现倾向于活化单核性细胞(巨嗜细胞、树状细胞和单核细胞)和NK细胞。带有优选的人基元的硫代磷酸酯CpG寡核苷酸也是单核性细胞和NK细胞的强活化子。
其它的稳定的核酸分子包括：非离子性DNA类似物，例如烷基或芳基的磷酸酯(其中带电的磷酸氧被烷基或芳基所取代)，磷酸二酯和烷基磷酸三酯，其中的带电的氧基团被烷基化。带有二醇例如四甘醇或六甘醇的核酸分子，已经显示在其一个终端或两个终端都是基本抵抗核酸酶的降解的。
术语“对象”指的是人或脊椎动物，包括狗、猫、马、牛、猪、绵羊、山羊、鸡、猴子、大鼠，和小鼠。
在本文中，术语“载体”指的是能够运送与之相连的其它核酸的核酸分子。优选的载体是能够对与之相连的核酸进行自我复制和表达的载体(例如，附加体)。能够对与之可工作地相连的基因的表达进行指导的载体被在本文中成为“表达载体”。总体来说，在重组DNA技术中有用的表达载体经常都是“质粒”形式的，其一般所指的是双链DNA的环形物，其在载体形式时不与染色体相连。在本说明书中，术语“质粒”和“载体”是可以相互替换的，因为质粒是最常用形式的载体。然而，本发明也包括其它形式的载体，即它们的功能等同物，以及随后变为本领域所知的那些载体。某些含有未甲基化CpG并具在在体内和体外的B细胞刺激活性的核酸
在根据随后的实施例1和2的描述所进行的对内源性逆病毒序列具有特异性的两种反义寡核苷酸刺激淋巴细胞效应的研究中，令人惊奇地发现了在24个“对照”(包括在“反义”ODN组中的各种杂乱的、有义的、和错配的对照)中的2个也能够介导B细胞活化和IgM分泌，而其它的“对照”则不具有这样的功能。
这两个研究的结果表明，这种“对照”ODN的B细胞活化机制可能不涉及反义效果，1)通过与基因库(GenBank)所列的脊椎动物DNA序列相比较，没有显示出比那些没有刺激性的ODN更多的同源性，以及2)这两个对照在使用了10μg的脾polyA+RNA的Northen印迹中没有显示出杂交。在不同的合成器上再次合成这些ODN，或用聚丙烯酰胺凝胶电泳或高压液相色谱来充分提纯，都得到相等的刺激，消除了杂质的可能性。采用来自C3H/HeJ小鼠的B细胞也见到了类似的刺激，这消除了脂多糖(LPS)污染会影响结果的可能性。
两个“对照”ODN所造成B细胞活化与两个“反义”ODN的情况相类似的事实提出了这样的可能性，即所有四个ODN对B细胞的刺激通过的是某种非反义机制进行的，所涉及的序列基元在所有的其它的非刺激性对照ODN中都不存在。比较这些序列发现，所有四个刺激性ODN含有的CpG二核苷酸处于与非刺激性对照不同的序列位置。
为了确定存在于刺激性ODN中的CpG基元是否负责所观察到的刺激作用，合成了超过300个的ODN，它们的长度范围是5-42个碱基对，含有甲基化或未甲基化的CpG二核苷酸，或者不含有CpG二核苷酸，并且位置也不同。这些ODN，包括两个原始的“对照”(即ODN1和2)以及两个原始的合成性“反义”物[ODN3D和3M；Krieg,A.M.J.Immunol.143:2448(1989)]，然后检查它们的脾细胞体外效果(代表性序列在表1中给出)。含有CpG二核苷酸的几种ODN诱导了B细胞的活化和IgM的分泌；这种刺激的程度一般可以由增加更多的CpG二核苷酸来提高(表1；比较ODN2与2a或3D与3Da和3Db)。看起来刺激不是反义机制或杂质的结果。ODN没有造成可检测的γδ或其它的T细胞种群的繁殖。
如果CpG二核苷酸发生突变，有丝分裂ODN序列都变为非刺激性的(表1；比较ODN1与1a；3D与3Dc；3M与3Ma；以及4与4a)，或如果CpG二核苷酸的胞嘧啶被5-甲基胞嘧啶取代(表1；ODN1b,2b,3Dd，和3Mb)。CpG基元的部分甲基化造成刺激效果的部分损失(比较2a与2c，表1)。相对照，对其它的胞嘧啶的甲基化没有减少ODN活性(ODN1c,2d,3De和3Mc)。这些数据确认了CpG基元存在于ODN中活化B细胞的重要元素。
在这些研究的过程中，变的更为清楚的是，在CpG二核苷酸侧翼的碱基在由ODN诱导的小鼠B细胞活化的确定中起着重要的作用。最佳的刺激基元被确定包括有以两个5′嘌呤(优选为GpA二核苷酸)和两个3′嘧啶(优选TpT或TpC二核苷酸)为侧翼的CpG。将CpG基元趋向靠近这种理想形态的ODN突变改善了刺激性(例如，表1，比较ODN2与2e；3M与3Md)，而那些打乱该基元的突变则减弱了刺激性(见表1，比较ODN 3D与3Df；4与4b,4c和4d)。另一方面，在CpG以外的突变没有减弱刺激性(例如，表1，比较ODN1与1d；3D与3Dg；3M与3Me)。对于活化人的细胞，最佳的侧翼碱基是稍有不同的(见表5)。
在那些实验过的ODNs中，长度小于8个碱基对的ODN都没有刺激性(例如，表1,ODN4e)。在所测试过的48个长度为8个碱基对的ODN中，确定了具有强刺激性的序列是TCAACGTT(ODN4)，其含有自我互补的“回文序列”AACGTT。在对该基元的进一步优化中，发现在两个终端都有G的ODN具有更强的刺激性，特别当ODN被硫代磷酸酯修饰了终端的核苷酸间的连接而具有对核酸酶的抗性后更是如此。ODN1585[5′GGGGTCAACGTTCAGGGGGG3′(SEQ ID NO:12)]中，前两个和最后的5个核苷酸间的连接都被硫代磷酸酯修饰了，使其在小鼠脾细胞的繁殖中平均提高了25.4倍，而相比之下，由ODN1638所造成的繁殖只增加了3.2倍，该ODN1638与ODN1585具有相同的序列，只是在两端的第10位的G被第10位的A所取代。富含G的终端的效果是其为顺式，对ODN加上多G的终端但不将CpG基元与1638一起加入细胞，也不会得到增强的繁殖。对于长度大于8个碱基对的核酸，含有未甲基化CpG的非回文序列基元被发现是更具有刺激性的。
含有6个碱基回文序列以及在5′端有TpC二核苷酸的10元ODN也是活性的(见表1,ODN4b,4c)。在5′端的其它的二核苷酸减弱刺激性(例如，ODN4f，对所有可能的16种二核苷酸都进行了测试)。有3′端二核苷酸存在并不足以补偿对5′端二核苷酸的缺失(见表1,ODN4g)。对回文序列打乱就消除了10元ODN的刺激性(例如，表1,ODN4h)，但是，在更长的ODN中，回文序列不是必须的。
表1：寡核苷酸对小鼠B细胞的刺激
                                                                                刺激性指数′ODN生产                            序列(5′至3′)                       3H尿嘧啶          IgM
 1(SEQ ID NO:13) GCTAGACGTTAGCGT     6.1±0.8  17.9±3.6
 1a(SEQ ID No:4) ■■■■■■T■■■■■■■■     1.2±0.2  1.7±0.5
 1b(SEQ ID No:14) ■■■■■■Z■■■■■■■■     1.2±0.1  1.8±0.0
 1c(SEQ ID No:15) ■■■■■■■■■■■■Z■■     10.3±4.4  9.5±1.8
 1d(SEQ ID No:16) ■■AT■■■■■■GAGC■     13.0±2.3  18.3±7.5
 2(SEQ ID No:17) ATGGAAGGTCCAGCGTTCTC     2.9±0.2  13.6±2.0
 2a(SEQ ID No:18) ■■C■■CTC■■G■■■■■■■■■  7.7±0.8  24.2±3.2
 2b(SEQ ID No:19) ■■Z■■CTC■ZG■■Z■■■■■■    1.6±0.5  2.8±2.2
 2c(SEQ ID No:20) ■■Z■■CTC■■G■■■■■■■■■    3.1±0.6  7.3±1.4
 2d(SEQ ID No:21) ■■C■■CTC■■G■■■■■■Z■■   7.4±1.4  27.7±5.4
 2e(SEQ ID No:22) ■■■■■■■■■■■■A■■■■■■ 5.6±2.0     ND
 3D(SEQ ID No:23) GAGAACGCTGGACCTTCCAT     4.9±0.5  19.9±3.6
 3Da(SEQ ID No:24) ■■■■■■■■■C■■■■■■■■■■ 6.6±1.5  33.9±6.8
 3Db(SEQ ID No:25) ■■■■■■■■■C■■■■■■■G■■ 10.1±2.8  25.4±0.8
 3Dc(SEQ ID No:26) ■■■C■A■■■■■■■■■■■■■■  1.0±0.1  1.2±0.5
 3Dd(SEQ ID No:27) ■■■■■Z■■■■■■■■■■■■■■ 1.2±0.2  1.0±0.4
 3De(SEQ ID No:28) ■■■■■■■■■■■■■Z■■■■■■ 4.4±1.2  18.8±4.4
 3Df(SEQ ID No:29) ■■■■■■■A■■■■■■■■■■■■ 1.6±0.1  7.7±0.4
 3Dg(SEQ ID No:30) ■■■■■■■■■CC■G■ACTG■■     6.1±1.5  18.6±1.5
 3M(SEQ ID No:31) TCCATGTCGGTCCTGATGCT     4.1±0.2  23.2±4.9
 3Ma(SEQ ID No:32) ■■■■■■CT■■■■■■■■■■■■ 0.9±0.1  1.8±0.5
 3Mb(SEQ ID No:33 ■■■■■■■Z■■■■■■■■■■■■ 1.3±0.3  1.5±0.6
 3Mc(SEQ ID No:34) ■■■■■■■■■■■Z■■■■■■■■ 5.4±1.5  8.5±2.6
 3Md(SEQ ID No:35) ■■■■■■A■■T■■■■■■■■■■ 17.2±9.4     ND
 3Me(SEQ ID No:36) ■■■■■■■■■■■■■■■C■■A■ 3.6±0.2  14.2±5.2
    4 TCAACGTT     6.1±1.4  19.2±5.2
    4a ■■■■GC■■     1.1±0.2  1.5±1.1
    4b ■■■GCGC■     4.5±0.2  9.6±3.4
    4c ■■■TCGA■     2.7±1.0     ND
    4d ■■TT■■AA     1.3±0.2     ND
    4e _■■■■■■■     1.3±0.2  1.1±0.5
    4f C■■■■■■■     3.9±1.4     ND
    4g __■■■■■■CT     1.4±0.3     ND
    4h ■■■■■■■C     1.2±0.2     ND
    LPS     7.8±2.5  4.8±1.0
′刺激指数是至少三个单独实验的平均值和标准偏差，并且与没有加入ODN的培养物的小孔进行比较。ND=未进行。CpG二核苷酸用下划线标出。黑点表示位置，短线表示缺失。Z代表5甲基胞嘧啶。
表2：确定对小鼠IL-6生产和B细胞活化为最优的CpG基元
                      IL-6(pg/ml)a
    ODN     序列(5′-3′)     SIb  IgM(ng/ml)c  CH12.LX  脾B细胞
512(SEQID No:31) TCCATGTCGGTCCTGATGCT 1300±106  627±43  5.8±0.3  7315±1324
1637(SEQID No:38) ■■■■■■C■■■■■■■■■■■■■ 136±27 46±6  1.7±0.2  770±72
1615(SEQID No:39) ■■■■■■G■■■■■■■■■■■■■ 1201±155 850±202  3.7±0.3  3212±617
1614(SEQID No:40) ■■■■■■A■■■■■■■■■■■■■ 1533±321 1812±103 10.8±0.6  7558±414
1636(SEQID No:41) ■■■■■■■■■A■■■■■■■■■■ 1181±76  947±132  5.4±0.4  3983±485
1634(SEQID No:42) ■■■■■■■■■C■■■■■■■■■■ 1049±223 1671±175 9.2±0.9  6256±261
1619(SEQID No:43) ■■■■■■■■■T■■■■■■■■■■ 1555±304 2908±129 12.5±1.0  8243±698
1618(SEQID No:44) ■■■■■■A■■T■■■■■■■■■■ 2109±291 2596±166  12.9±0.7  10425±674
1639(SEQID No:45) ■■■■■AA■■T■■■■■■■■■■ 1827±83 202±132  11.5±0.4  9489±103
1707(SEQID No:46) ■■■■■■A■■TC■■■■■■■■■ ND  1147±175  4.0±0.2  3534±217
1708(SEQID No:47) ■■■■■CA■■TG■■■■■■■■  ND  59±3  1.5±0.1  466±109
黑点代表位置；CpG二核苷酸用下划线标出；ND=未进行a进行的实验为至少三次近似的结果。CH12.LX和脾B细胞的未刺激的对照培养物的IL-6水平≤10pg/ml。未刺激的培养物的IgM水平为547±82ng/ml。CpG二核苷酸用下划线标出，黑点代表位置。b[3H]尿嘧啶吸收用对未刺激的对照(2322.67±213.68cpm)所提高的倍数表示(SI：刺激指数)。细胞用20μM各种CpG O-ODN刺激。数据代表三个数据的平均值±标准偏差。c用ELISA测定。
对淋巴细胞活化的动力学的研究采用了小鼠脾细胞。在加入ODN的同时对细胞进行冲击，4小时后收获，已经有了2倍的3H尿嘧啶的吸收。刺激的峰值在12-48小时，然后降低。24小时后，检测不到完整的ODN，这也许就是当纯化的B细胞随后在有或没有抗-IgM(亚促有丝分裂剂量)条件下与CpG ODN一起培养时发生刺激性下降的原因，在48小时后，两种促有丝分裂还造成繁殖也同时增强约10倍。刺激的力度依赖于浓度，并且在对两种物质为优化的条件下都一致地超过LPS。含有抗核酸酶的硫代磷酸酯主链的寡核苷酸比未修饰的寡核苷酸强大约200倍。
采用细胞生长循环来确定由CpG-ODN活化的B细胞的数量。CpG-ODN诱导了在大于95％的B细胞的循环。用流式细胞仪区分脾B淋巴细胞为CD23-(边缘区)和CD23+(小泡区)亚群，它们对ODN诱导的刺激都有同样的反应，用Percoll梯度区分的B细胞类群也是休眠和活化的种群。这些研究表明，CpG-ODN诱导几乎所有的B细胞进入细胞循环。免疫刺激性核酸分子对小鼠B细胞编程性细胞死亡的封闭
有些B细胞系，例如WEHI-231，被诱导经历生长的休眠和/或编程性细胞死亡，应答于它们的抗原受体与抗-IgM的交联[Jakway,J.P.et al.,″Growth regulation ofthe B lymphoma cell lineWEHI-231 by anti-immunoglobulin,lipopolysaccharide and otherbacterial products″J.Immunol.137:2225(1986)；Tsubata,T.,J.Wuand T.Honio:B-cell apoptosis indueced by antigen receptorcorsslinking is blocked by a T-cell signal through CD40.″Nature364:645(1993)]。用某些刺激物如LPS和CD40配体可以将WEHI-231细胞从这种生长休眠状态下恢复出来。含有CpG基元的ODN也被发现可以保护WEHI-231细胞不受抗-IgM诱导的生长休眠的影响，表明这种效果不需要辅助细胞种群。随后的研究工作表明，CpG ODN诱导Bcl-x和myc表达，它们可能对抵抗编程性细胞死亡有作用。再者，CpG核酸被发现可以封闭人细胞的编程性细胞死亡。这种对编程性细胞死亡的抑制具有重要性，因为它将提高和延长CpG DNA的免疫活化。鉴定对诱导小鼠IL-6和IgM分泌以及B细胞繁殖为最优化的CpG基元
为了确定最优化的B细胞刺激性CpG基元是否与对IL-6分泌的最优化CpG基元相同，研究了一组ODN，它们的CpG二核苷酸的侧翼碱基被逐步取代。对该组ODN的研究着重于分析它们对B细胞繁殖、Ig生产、IL-6分泌的效应，使用了脾B细胞和CH12.LX细胞。如表2所示，优化的刺激性基元含有未甲基化的CpG，侧翼为两个5′嘌呤和两个3′嘧啶。一般地说，5′嘌呤向3′嘧啶的突变和3′嘧啶向嘌呤的突变都显著地降低这种效应。从5′嘌呤向C的变化是特别的有破坏性的，但是，从5′嘌呤向T的变化或3′嘧啶向嘌呤的变化所产生的影响就比较小。根据这些分析以及其它的ODN的结果，确定了优化的CpG基元对诱导IL-6分泌为TGACGTT，其相同于优化的促有丝分裂和IgM诱导性CpG基元(表2)。该基元比任何所研究过的含有回文序列的序列都更具有刺激性(1639,1707和1708)。由在细菌DNA中或寡核苷酸中的CpG基元诱导的小鼠细胞因子的分泌
如在实施例9中所描述的那样，以CpG DNA刺激后的脾细胞分泌的IL-6的数量用ELISA测定。采用的不是全脾细胞，而是用去除了T细胞的脾细胞培养物，进行了体外研究，经过最初的研究发现，T细胞对CpG DNA刺激的脾细胞生产IL-6没有任何作用。如表3所示，用大肠杆菌DNA培养的细胞明显地提高了IL-6的生产，而用小牛胸腺DNA培养的细胞则没有提高。为了确认所观察到的用大肠杆菌DNA培养的细胞的IL-6生产的提高不是由于污染物或其它的细菌产物所造成的，在进行分析之前，对DNA用DNA酶进行处理。这种DNA酶的预处理取消了由大肠杆菌DNA诱导的IL-6的生产(表3)。此外，从LPS-非应答C3H/HeJ小鼠来的脾细胞应答了细菌DNA而产生了相似水平的IL-6。为了研究由大肠杆菌DNA诱导的IL-6的分泌是否由细菌DNA中的未甲基化CpG二核苷酸所介导，检验了甲基化的大肠杆菌DNA和一组合成性ODN。如表3所示，CpG ODN显著地诱导了IL-6的分泌(ODN5a,5b,5c)，而CpG甲基化大肠杆菌DNA，或含有甲基化CpG的ODN(ODN5f)，或不含CpG的ODN(ODN5d)则都没有发生诱导。在CpG二核苷酸(ODN5b)以外位置的改变，以及其它的胞嘧啶的甲基化(ODN5g)都没有减弱CpG ODN的效应。在含有三个CpG的ODN中对一个CpG甲基化的结果是部分地降低了其刺激性(表3，比较ODN5c和5e)。
表3：由在细菌DNA中或寡核苷酸中的CpG基元诱导的小
鼠IL-6的分泌
    处理     IL-6(pg/ml)
小牛胸腺DNA     ≤10
小牛胸腺DNA+DNase     ≤10
大肠杆菌DNA  1169.5±94.1
大肠杆菌DNA+DNase     ≤10
CpG甲基化大肠杆菌DNA     ≤10
    LPS  280.1±17.1
培养基(没有DNA)     ≤10
    ODN
 5a SEQ ID NO:1 ATGGACTCTCCAGCGTTCTC  1096.4±372.0
 5b SEQ ID NO:2 ■■■■AGG■■■■■A■■■■■■■■ 1124.5±126.2
 5c SEQ ID NO:3 ■■C■■■■■■■G■■■■■■■ 1783.0±189.5
 5d SEQ ID NO:4 ■■■■AGG■■C■■T■■■■■■■ ≤10
 5e SEQ ID NO:5 ■■C■■■■■■■G■■Z■■■■■■ 851.1±114.4
 5f SEQ ID NO:6 ■■Z■■■■■■ZG■■Z■■■■■■ ≤10
 5g SEQ ID NO:7 ■■C■■■■■■■G■■■■■■Z■■ 1862.3±87.26
去除了T细胞的来自DBA/2小鼠的脾细胞用磷酸二酯修饰的寡核苷酸(O-ODN)(20μM)、小牛胸腺DNA(50μg/ml)、经过或不经过酶处理的大肠杆菌DNA(50μg/ml)、或LPS(10μg/ml)分别刺激24小时。数据代表三个数据的平均值±SD。CpG二核苷酸用下划线标出，黑点代表位置。Z表示5-甲基胞嘧啶。CpG基元可以用作人工附剂
免疫应答的非特异性刺激物是已知的附剂。使用附剂对诱导相对于抗原的强抗体应答是重要的(Harlow and Lane,Antibodies:A Laboratory manual,Cold Spring harbor,N.Y.CurrentEdition；通过在此引述而合并于本文)。附剂总体效应是强烈的，它们的重要性是不会被过分强调的。附剂的作用允许用更小剂量的抗原来产生更为一致的抗体应答。对免疫应答的非特异性活化经常可以分辨出获得免疫应答的成功或失败。应该在第一次注射的时候使用附剂，除非有非常特殊的理由来避免这样做。大多数的附剂含有两个成分。一个成分是用来保护抗原不被快速分解代谢[例如，脂质体或合成的表面活性剂(Hunter et al.,1981)]。脂质体仅仅在免疫原被整合到外脂层时有效，内埋的分子是免疫系统看不见的。其它的成分则是非特异性刺激免疫应答的物质。这些物质的作用是提高淋巴因子的水平。淋巴因子直接刺激抗原处理细胞的活性，并引起注射位置的局部发炎反应。早期的工作完全依赖于热杀死细菌(Dienes1936)或脂多糖(LPS)(Johnsonet al.,1956)。LPS是合理的毒性的，通过对它的结构的分析，其作为附剂的大多数的性质已经被证实为在于称作脂质A的部分。脂质A可以由多种合成的和天然的形式获得，其毒性远远小于LPS，但仍然保存了其前身的LPS分子的大多数良好的附剂性质。脂质A化合物经常用脂质体提供。
近来，寻找强有力的并带有更可接受的副作用的附剂的工作导致了新的合成附剂的产生。在本发明中，我们提供了序列第1826号，即TCCATGACGTTCCTGACGTT(SEQ ID NO:10)，它是一种包括了含CpG的核酸的附剂。该序列是强免疫活化序列，并且是非常优秀的附剂，其效率相等于完全Freund′s，甚至超过该附剂，但是，又没有明显的毒性。CpG基元诱导小鼠IL-6生产的效价
细菌DNA和CpG ODN在去除了T细胞的小鼠脾细胞中诱导的IL-6生产是属于剂量依赖型的，但是，脊椎动物DNA和非CpG ODN则不是(图1)。IL-6的生产在大约50μg/ml细菌DNA和40μM的CpG O-ODN水平时是平台式的。由细菌DNA和CpGODN诱导的IL-6的生产的最大值分别在1-1.5ng/ml和2-4ng/ml。这些水平比在LPS(0.35ng/ml)刺激后所见到的要明显地高(图1A)。为了评价带有核酸酶抗性DNA主链的CpG ODN是否也可以诱导IL-6的生产，向去除了T细胞的小鼠脾细胞中加入S-ODN。CpG S-ODN也诱导了剂量依赖型的IL-6生产，并且水平也类似于CpG O-ODN，而非CpG S-ODN则没有诱导IL-6的生产(图1C)。浓度为0.05μM的CpG S-ODN可以在这些细胞中诱导最大的IL-6生产。这个结果表明核酸酶抗性DNA主链修饰保留的CpG DNA对IL-6生产的序列特异性能力，并且CpG S-ODN在该检测系统中比CpG ODN的强度高出80倍以上。CpG DNA在体内对小鼠IL-6分泌的诱导
为了评价细菌DNA和CpG S-ODN体内诱导IL-6分泌的能力，给BALB/c小鼠静脉注射100μg大肠杆菌DNA、小牛胸腺DNA、CpG或非刺激性S-ODN,2小时后采血。在注射了大肠杆菌DNA的组中，血清内IL-6水平为约13ng/ml，而在注射了小牛胸腺DAN的组和注射了PBS的组中，血清内没有检测到IL-6(表4)。在注射了CpG S-ODN的组中，血清内IL-6的水平为约20ng/ml。在注射了非刺激性S-ODN的组中，没有检测到IL-6(表4)。
表4．CpG DAN刺激诱导的小鼠体内IL-6分泌
       刺激物     IL-6(pg/ml)
        PBS        ＜50
    大肠杆菌DNA     13858±3143
    小牛胸腺DNA        ＜50
    CpG S-ODN     20715±606
    非-CpG S-ODN        ＜50
小鼠(每组2只)接受静脉注射100μl的PBS、200μg的大肠杆菌DNA或小牛胸腺DNA、或500μg的CpG S-ODN或非-CpG对照S-ODN。注射后2小时采血，按1∶10稀释，每种血清用ELISA测定IL-6。ELISA测定IL-6的敏感限定为5pg/ml。CpG S-ODN的序列是5′GCATGACGTTGAGCT3′(SEQ ID NO:48)，非刺激性S-ODN的序列是5′TCTAGATGTTAGCGT3′(SEQID NO:49)。应该注意的是，虽然在序列48中有CpG，但对于刺激效应来讲它太靠近3′端了。数据代表两个结果的平均值±标准偏差。实验进行至少两次且结果类似。用CpG基元刺激后小鼠体内分泌IL-6的动力学
为了评价CpG DNA在体内诱导IL-6分泌的动力学，对BALB/c小鼠静脉注射CpG或对照非-CpG S-ODN。在注射了CpGS-ODN的组中，血清IL-6水平在1小时内就明显提高，并且在2小时达到峰值水平约9ng/ml(图2)。血清中IL-6蛋白质水平在4小时后明显下降，并在12小时后回到基础水平。与CpG DNA刺激的组相反，在注射了非刺激性S-ODN或PBS的组中，血清内IL-6没有明显提高(图2)。CpG基元在体内诱导的IL-6mRNA表达的动力学和组织分布
如图2所示，在CpG DAN刺激后，血清IL-6水平迅速提高。为了调查这种血清IL-6的可能的组织起源以及CpG DNA刺激后体内IL-6基因表达的动力学，在刺激后的不同的时间点对BALB/c小鼠静脉内注射CpG或非CpG S-ODN和RNA,RNA分别取自肝、脾、胸腺、和骨髓。如图3A所示，注射CpG S-ODN后30分钟内，在肝、脾、和胸腺的IL-6mRNA水平已经明显提高。肝内IL-6mRNA在注射后2小时达到峰值，随后迅速下降并在刺激后8小时回到基础水平(图3A)。脾内IL-6mRNA在刺激后2小时达到峰值，然后逐渐降低(图3A)。胸腺内IL-6mRNA在注射后1小时达到峰值，然后逐渐降低(图3A)。在注射CpG S-ODN后1小时内，骨髓内IL-6mRAN明显提高，然后恢复到基础水平。作为对CpG S-ODN的应答，肝、脾、和胸腺的IL-6mRNA表达的提高明显高于骨髓。CpG DNA诱导的小鼠细胞因子表达的形式
在体内或在全脾细胞中，在最初的6个小时内，下列的白细胞介素如，IL-2、IL-3、IL-4、IL-5、和IL-10等都没有检测到其蛋白质水平的显著提高[Ilinman,D.M.et al.,(1996)Proc.Natl.Acad.Sci.USA93:2879-2883]。然而，在注射CpG ODN的小鼠血清中，TNF-α的水平在30分钟内明显提高，IL-6的水平在2小时内显著地升高。在脾细胞中，在首先的2小时内也检测到IL-12和伽玛干扰素(IFN-γ)mRNA的表达的提高。
表5．CpG寡核苷酸诱导人PBMC细胞因子的分泌
    ODN       序列(5′-3′)  IL-61 TNF-α1 IFN-γ1  GM-CSF  IL-12
512SEQ ID NO:31  TCCATGTCGGTCCTGATGCT 500 140 15.6 70 250
1637SEQ ID NO:38 ■■■■■■C■■■■■■■■■■■■■ 550 16 7.8 15.6 16
1615SEQ ID NO:39 ■■■■■■G■■■■■■■■■■■■■ 600 145 7.8 45 145
1614SEQ ID NO:40 ■■■■■■A■■■■■■■■■■■■■ 550 31 0 50 31
1636SEQ ID NO:41 ■■■■■■■■■A■■■■■■■■■■ 325 250 35 40 250
1634SEQ ID NO:42 ■■■■■■■■■C■■■■■■■■■■ 300 400 40 85 400
1619SEQ ID NO:43 ■■■■■■■■■T■■■■■■■■■■ 275 450 200 80 450
1618SEQ IDNO:44 ■■■■■■A■■T■■■■■■■■■■ 300 60 15.6 15.6 62
1639SEQ ID NO:45 ■■■■■AA■■T■■■■■■■■■■ 625 220 15.6 40 220
1707SEQ ID NOL:46 ■■■■■■A■■TC■■■■■■■■■ 300 70 17 0 70
1708SEQ ID NO:47 ■■■■■CA■■TG■■■■■■■■■ 270 10 17 ND 10
黑点代表位置，CpG二核苷酸用下划线标出。
1由ELISA测定，采用了从R&D System的Quantikine试剂盒(pg/ml)。在收集上清液和检测前，细胞培养在10％自身血清并加以给出剂量的寡脱氧核苷酸(12μg/ml)，对TNF-α为4小时，对其它的细胞因子为24小时。数据代表相对于没有加入寡脱氧核苷酸的小孔的细胞因子的提高水平。CpG DNA诱导的特异性单核细胞人PBMC的细胞因子的分泌
采用了与研究小鼠细胞因子表达的同一组ODN，来确定人的细胞是否也被CpG基元诱导表达细胞因子(或繁殖)，并确定有关的CpG基元。寡核苷酸1619(GTCGTT)是对TNF-α和INF-γ的最好的诱导物，与其几乎相等的在寡核苷酸1634中的基元(GTCGCT)则近随其后(表5)。在寡脱氧核苷酸1637和1614中的基元(GCCGGT和GACGGT)导致强烈的IL-6分泌但对其它的细胞因子则相对没有诱导。因此，这就显示人淋巴细胞同小鼠淋巴细胞一样，应答于CpG二核苷酸而分泌不同的细胞因子，具体由其周围的碱基所决定。然而，对刺激小鼠基元为最好的基元则不同于对人细胞最有效的基元。某些CpG寡脱氧核苷酸对活化人细胞是很差的(寡脱氧核苷酸1707、1708，它们分别含有形成回文的序列GACGTC和CACGTG)。
对DNA应答的细胞表现为单核细胞，因为用L-亮氨酰-亮氨酸甲基酯(L-LME)处理可以消除细胞因子的分泌，该物质对单核细胞为选择性毒性物(但是，也对T淋巴细胞和NK细胞为细胞毒性的)，并不影响B细胞的Ig分泌(表6)。经过L-LME处理而存活的细胞用锥虫蓝排斥有＞95％的存活率，这表明，这些细胞缺少细胞因子应答并不简单反映非特异性的细胞死亡。应答大肠杆菌DNA的细胞因子分泌需要未甲基化CpG基元，因为甲基化消除了大肠杆菌DNA的作用(表6中倒数第二行)。LPS对DNA的污染并不能解释这些结果，因为在天然的和甲基化的DNA中的污染是相同水平的，而且，加入两倍最高水平的污染物LPS也没有产生效应(未示出)。
表6．CpG DNA诱导入PBMC的细胞因子的分泌
            DNA    TNF-α(pg/ml)1   IL-6(pg/ml)   IFN-γ(pg/ml)  RANTES(pg/ml)
       EC DNA(50μg/ml)     900   12,000     700    1560
       EC DNA(5μg/ml)     850   11,000     400    750
       EC DNA(0.5μg/ml)     500     ND     200     0
       EC DNA(0.05μg/ml)    62.5   10,000     15.6     0
    EC DNA(50μg/ml)+L-LME2     0     ND     ND     ND
    EC DNA(10μg/ml)Methyl3     0     5     ND     ND
      CT DNA(50μg/ml)     0    600     0     0
1所有细胞因子的水平的测定都用ELISA进行，采用的是R&D Systems的Quantikine试剂盒，如同前面的表中描述的一样。结果代表从不同的供体来的PBMC的情况。
2对细胞用L-亮氨酰-亮氨酸甲基酯(M-LME)预处理15分钟，来确定在这样的条件下的细胞因子的产生是否是从单核细胞中来的(或来自于其它的对L-LME敏感的细胞)。
3对大肠杆菌DNA用2U/μgDNA的CpG甲基酶(NewEngland Biolabs)甲基化，以HpA-Ⅱ和Msp-Ⅰ酶解来确认甲基化。作为阴性对照，样品内包括的污染物LPS量为在该实验条件下没有诱导可检测细胞因子生产的最高量大肠杆菌DNA中的污染物的两倍。
ND=未进行。
用L-LME处理的PBMC损失了细胞因子生产这个现象建议单核细胞也许对应答CpG DNA的细胞因子生产负责。为了更直接地验证这个假设，测试了CpG DNA对高纯度的人单核细胞和巨嗜细胞的效果。如同所假设的那样，CpG DNA直接活化人巨嗜细胞生产IL-6、GM-CSF、和TNF-α，而非CpG DNA则不能(表7)。
表7.CpG DNA诱导纯化人巨嗜细胞分泌细胞因子
 IL-6(pg/ml)    GM-CSF(pg/ml)   TNF-α(pg/ml)
    细胞     0        0       0
 CT DNA(50μg/ml)     0        0       0
 EC DNA(50μg/ml)     2000        15.6       1000
应答CpG基元的诱导的小鼠IgM生产中的IL-6的生物学作用
上述的动力学研究揭示了在CpG刺激后1小时诱导了IL-6的分泌并进而有IgM的分泌。因为对ODN诱导IL-6分泌的优化CpG基元相同于IgM的优化基元(表2)，所以检测了CpG基元是否单独诱导IgM和IL-6生产，以及是否IgM的生产依赖于IL-6的产生。加入中性抗-IL-6抗体抑制了体外的由CpG ODN介导的剂量依赖型的IgM生产，但是对照抗体则不能(图4A)。相对照的是，加入IL-6则没有影响CpG诱导的B细胞的繁殖水平和其基础水平(图4B)。应答CpG DNA的IL-6启动子的转录活性的提高
用CpG DNA刺激后的IL-6mRNA和蛋白质水平的提高可以是来源于转录调节或转录后调节的结果。为了确定IL-6启动子的转录活性是否在与CpG ODN一起培养的B细胞中被上调，采用了应答CpG DNA产生IL-6的小鼠B细胞系wEHI-231，对起用IL-6启动子CAT构建物转染[pIL-6/CAT(Pottratz,S.T.et al.,17B-estradiol)Inhibits expression of human interleuking-6-promoter-reporter constructs by a receptor-dependent mechanism.J.Clin.Invest.93:944]。CAT检测在用各种浓度的CpG或非CpGODN刺激后进行。如图5所示，CpG ODN诱导的CAT活性的提高是剂量依赖型的，而非CpG ODN则没有诱导CAT活性。这就肯定了CpG诱导IL-6启动子的转录活性。CpG ODN诱导的B细胞活化对5′和3′硫代磷酸酯核苷酸内连接数目的依赖
为了确定ODN主链上的部分的硫修饰是否足以提高B细胞的活化，选取了一系列具有相同的序列但在5′和3′端的核苷酸内连接的S数目不同的ODN，并测定了它们的效果。根据以前的对核酸酶降解ODN的研究，确定了需要在ODN的5′端有至少两个硫代磷酸酯连接才可以保护ODN不被细胞性外切核酸酶或内切核酸酶所降解。因此，只对含有两个5′硫代磷酸酯修饰的连接的嵌合性ODN以及各种数目的3′修饰的连接进行了检测。
试验了这些ODN对淋巴细胞的刺激效果，使用了三种浓度(3.3,10，和30μM)，测定了它们在处理过的脾细胞培养物中的总RNA合成水平(由3H尿苷整合)或DNA合成(有3H胸苷整合)水平(实施例10)。带有CpG基元的O-ODN(0/0硫代磷酸酯修饰)对脾细胞没有刺激，除非在加入的培养基中的浓度为至少10μM(实施例10)。然而，当这个序列发生了在5′端的两个S连接修饰和在3′端的至少3个S连接的修饰时，则在3.3μM的剂量时有显著的刺激表现出来。在这种低剂量的条件下，随着3′端修饰的碱基数目的增加有刺激水平的逐渐增加，当达到或超过6个修饰时，刺激指数开始下降。一般地讲，对脾细胞刺激的优化的3′的S修饰数目为5。在所有测试的3种浓度中，S-ODN都比嵌合性化合物的刺激性小。CpG介导的淋巴细胞活化对主链修饰类型的依赖
硫代磷酸酯修饰的ODN(S-ODN)比磷酸二酯修饰的ODN(O-ODN)具有更强的对核酸酶的抗性。因此，由S-ODN和S-O-ODN(即，嵌合性硫代磷酸酯ODN，其中的中央连接是磷酸二酯，但有2个5′和5个3′的连接是硫代磷酸酯修饰的)所造成的比O-ODN更高的免疫刺激可能是前者的对核酸酶的抗性的结果。为了确定ODN核酸酶抗性在CpG ODN的免疫刺激中的作用，测试了嵌合性ODN的刺激效果，这些ODN中的5′端和3′端都附带有核酸酶抗性，来自于甲基磷酸酯(MP-)、甲基硫代磷酸酯(MPS-)、磷酸二酯(S-)、或二硫代磷酸酯(S2-)的核苷酸内的连接(实施例10)。这些研究显示，无论它们的核酸酶抗性如何，MP-O-ODN实际上都比O-ODN的免疫刺激性低。然而，通过用5′端和3′端的MPS核苷酸内连接来取代非桥连O分子将MP和S的修饰结合起来，就使免疫刺激的水平恢复到稍微高于O-ODN的水平。
S-O-ODN比O-ODN更为具有刺激性，甚至比S-ODN更具有刺激性，至少在高于3.3μM浓度时如此。在低于3μM浓度时，带有3M序列的S-ODN比相应的S-O-ODN更具刺激性，而带有3D序列的S-ODN则比相应的S-O-ODN的刺激性弱(实施例10)。在比较这两个序列的刺激性CpG基元时，注意到了3D序列与侧翼为2个5′端嘌呤和2个3′端嘧啶的CpG这样的基元完全吻合。然而，在ODN3D中紧靠CpG的侧翼碱基不是优化的，其具有5′端嘧啶和3′端嘌呤。根据进一步的试验，发现免疫刺激所需要的序列对S-ODN比对S-O-ODN和O-ODN更为严谨。S-ODN与优化的CpG基元吻合性差就会对淋巴细胞的活化没有刺激或很小(例如，序列3D)。然而，与基元吻合很好的S-ODN，特别是在紧靠CpG的侧翼位置，都比相应的S-O-ODN更具有刺激性(例如，序列3M，序列4和6)，虽然在高浓度(大于3μM)时的峰值效应是S-O-ODN的更强(实施例10)。
S2-O-ODN具有显著的刺激性，并且在每个测试浓度都引起比相应的S-ODN或S-O-ODN更为强烈的对淋巴细胞的活化。
在带有S或S2取代的CpG ODN中所见到的对B细胞刺激的升高可能是下述的一个或全部的效应的结果：核酸酶抗性、增强的细胞吸收、增强的蛋白质的结合、和改变了的细胞间的定位。然而，核酸酶抗性不会是唯一的解释，因为MP-O-ODN实际上比带有CpG基元的O-ODN的刺激性低。以前的研究已经显示淋巴细胞对ODN的吸收明显地受到主链的化学结构的影响[Zhao et al.,(1993)Comparison of cellular binding and uptake ofantisense phosphodiester,phosphorothioate,and mixedphosphorethioate and methylphosphonate olignucleotides.(Andisense Research and Development3,53-66；Zhao et al.,(1994)Stage specific oligonucleotide uptake in murine bone marrow B cellprecursors.Blood 84,3660-36666.)]。细胞膜结合和吸收的最高值见于S-ODN，随后是S-O-ODN,O-ODN，和MP-ODN。这种吸收的区分与免疫刺激的程度正好相关。含有未甲基化CpG的寡核苷酸具有时NK细胞的刺激活性
为了确定含有CpG的寡核苷酸除了刺激B细胞外是否还对天然杀死细胞(NK)具有刺激活性，进行了各种实验。如表8所示，用CpG ODN1和3Dd培养的脾细胞中有明显的NK活性的诱导。相比之下，用非CpG对照ODN处理的效应物都相对没有诱导。
表8.CpG寡核苷酸(ODN)诱导的NK活性
   ％YAC-1特异性溶胞*      ％2C11特异性溶胞
   效应物     靶标    效应物     靶标
    ODN     50∶1     100∶1     50∶1     100∶1
    无     -1.1     -1.4     15.3     16.6
    1     16.1     24.5     38.7     47.2
    3Dd     17.1     27.0     37.0     40.0
非-CpG ODN     -1.6     -1.7     14.8     15.4
由含有CpG基元的DNA而不是非CpG DNA诱导的NK活性
在37℃下对细菌DNA培养18小时，然后测定杀死K562(人)或Yac-1(小鼠)靶细胞在去除了B细胞的脾细胞中和在人PBMC细胞中诱导的溶胞活性，但是，脊椎动物的DNA没有这种效果(表9)。为了确定细菌DNA的刺激活性是否归因于其未甲基化CpG二核苷酸水平的提高，对含有未甲基化CpG二核苷酸、甲基化的CpG二核苷酸、或没有CpG二核苷酸的50个以上的合成性ODN进行了测验。结果如表9所示，显示了合成的ODN可以显著地刺激NK活性，只要它们含有至少一个未甲基化CpG二核苷酸即可。在CpG处于回文序列中的ODN(例如ODN1585，其含有回文序列AACGTT)与没有回文序列的ODN(例如ODN1613或1619)之间没有观察到明显的刺激性的区别，要注意的是，优化的刺激一般见于那些CpG侧翼为2个5′端嘌呤或5′端GpT二核苷酸和2个3′端嘧啶的ODN中。动力学实验表明NK活性的峰值出现在加入OND后的约18小时处。数据指出小鼠NK应答依赖于在先的CpG DNA对单核细胞的活化，导致的IL-12、THF-α、和IFN-α/b的生产(实施例11)。表9．含有CpG基元的DNA但不是非CpG DNA诱导的NK活性
          LU/106
加入的DNA或细胞因子   小鼠细胞    人细胞
实验1 无     0.00     0.00
IL-2     16.68     15.82
大肠杆菌DNA     7.23     5.05
小牛胸腺DNA     0.00     0.00
实验2 无     0.00     3.28
1585ggGGTCAACGTTGACgggg (SEQ ID NO.12)     7.38     17.98
1629-------------gtc------------- (SEQ ID NO.50)  0.00     4.4
实验3 无     0.00
1613GCTAGACGTTAGTGT (SEQ ID NO.51)     5.22
1769------------Z------------ (SEQ ID NO.52)     0.02      ND
1619TCCATGTCGTTCCTGATGCT (SEQ ID NO.43)     3.35
1765-----------Z----------- (SEQ ID NO.53)     0.11
在ODN中的CpG二核苷酸用下划线标出；Z代表甲基胞嘧啶。小写字母代表核酸酶抗性硫代磷酸酯修饰的核苷酸内连接，其在效价实验中显示了比非修饰的ODN强20倍以上的力度，具体根据侧翼碱基来决定。多G结尾(g)用在了某些ODN中，因为它们显著地提高了ODN吸收水平。
从所有这些研究中，得到了对CpG DNA的免疫效应的更为全面的理解，并将其总结于图6中。
CpG基元的免疫活化可能依赖于CpG侧翼的碱基，在ODN中的CpG的数量和间隔。虽然在理想的碱基成分中的单一CpG可以很强并且是有用的免疫活化子，但在含有几个CpG并带有适宜的侧翼碱基和间隔的ODN中能见到更为优越的效果。对B细胞的活化来讲，优化的CpG基元是TGACGTT。
下面所进行的研究是为了确定对刺激人细胞为优化的ODN序列，检验了变换CpG二核苷酸的数目、间隔和侧翼碱基的效果。确定带有对活化人NK细胞为优化的CpG基元的硫代磷酸酯ODN
要想应用于临床，则ODN必须以不受核酸酶降解的形式提供给对象。用磷酸二酯ODN来达到这个目的的方法是本领域所熟知的，包括将其包被在脂质中或在释放系统中，例如，在纳米颗粒中。这种保护还可以利用对DNA的化学修饰来达到，例如，修饰的DNA主链包括那些核苷酸间连接为抗核酸酶的。有些修饰可以赋予另外的所需性质，例如提高细胞吸收。磷酸二酯连接可以通过用硫替代非桥连氧原子中的一个来进行修饰，从而得到硫代磷酸酯DNA。如果硫代磷酸酯DNA具有CpG基元，则其具有提高了的细胞吸收(Krieg et al.,Antisense Res.Dev.6:133,1996)和改善了的B细胞刺激性。因为NK活化相关于体内的附剂效应，所以确定那些可以活化人NK细胞的硫代磷酸酯ODN是十分重要的。
检验了不同的硫代磷酸酯ODNs-即在各种碱基成分中含有CpG二核苷酸-对人NK细胞活化的效果(表10)。ODN1840含有2份TGTCGTT基元，具有明显的NK溶胞活性(表10)。为了进一步确定其它的对NK活化为优化的ODN，对大约100个含有不同数量和间隔的CpG基元的ODN进行了检测，并以ODN1982作为对照。结果列于表11。
有效的ODNs以TC或TG作为5′端开始，然而，这个要求不是必须的。带有内含CpG基元的ODNs(例如，ODN1840)一般是比GTCGCT基元紧靠5′端的ODN较弱的刺激物(例如，ODN1967和1968)。ODN1968在其3′端的那一半具有第二个GTCGTT基元，其始终比不含第二个基元的ODN1967更具有刺激性。然而，ODN1967比实验1和3中比ODN1968稍强，在实验2中则不是。ODN2005具有第三个GTCGTT基元，其所诱导的NK活性平均比1968稍高。然而，ODN2006中GTCGTT基元之间的间隔由于在每个基元之间都加入了两个T而被增加了，其比ODN2005和ODN2007更为优越，后者只有一个基元添加了间隔性的2个T。在CpG基元之间的最小可接受的间隔是一个核苷酸，只要该ODN在3′端有两个嘧啶(优选T)即可(如ODN2015)。令人惊奇的是，用5′端的T来把两个GTCGTT基元对头连接起来也创造了对NK活性为合理强度的诱导物(例如，ODN2016)。选择胸腺嘧啶(T)来分开相连的CpG二核苷酸不是绝对的，因为ODN2002诱导的相当的NK活化，虽然其中是由腺嘌呤(A)将其CpGs(即CGACGTT)间隔开来。还应该注意的是，不含CpG的ODN(例如ODN1982)，含有成串的CpGs的ODN，以及CpGs存在于坏的序列中的ODN(例如，ODN2010)都没有对NK活化的刺激效应。
表10.ODN诱导NK溶胞活性(LU)
    ODN     序列(5′-3′)     LU
  细胞自己    0.01
    1754  ACCATGGACGATCTGTTTCCCCTC    0.02
    1758  TCTCCCAGCGTGCGCCAT    0.05
    1761  TACCGCGTGCGACCCTCT    0.05
    1776  ACCATGGACGAACTGTTTCCCCTC    0.03
    1777  ACCATGGACGAGCTGTTTCCCCTC    0.05
    1778  ACCATGGACGACCTGTTTCCCCTC    0.01
    1779  ACCATGGACGTACTGTTTCCCCTC    0.02
    1780  ACCATGGACGGTCTGTTTCCCCTC    0.29
    1781  ACCATGGACGTTCTGTTTCCCCTC    0.38
    1823  GCATGACGTTGAGCT    0.08
    1824  CACGTTGAGGGGCAT    0.01
    1825  CTGCTGAGACTGGAG    0.01
    1828  TCAGCGTGCGCC    0.01
    1829  ATGACGTTCCTGACGTT    0.42
    18302  随机序列    0.25
    1834  TCTCCCAGCGGGCGCAT    0.00
    1836  TCTCCCAGCGCGCGCCAT    0.46
    1840  TCCATGTCGTTCCTGTCGTT    2.70
    1841  TCCATAGCGTTCCTAGCGTT    1.45
    1842  TCGTCGCTGTCTCCGCTTCTT    0.06
    1851  TCCTGACGTTCCTGACGTT    2.32
1溶胞单位(LU)按照所述(8)进行。简言之，从正常的供体收集PBMC，用Ficoll离心，然后采用或不采用给定剂量的ODN(加入的剂量为6μg/ml)培养24小时。确定它们的对51Cr-标记的K562细胞的溶胞能力。给出的结果是从几个不同的正常人供体获得的典型的结果。2该寡核苷酸混合物含有的是在每个位置上从4种碱基中随机选择的碱基。
表11．带有良好基元的硫代磷酸酯CpG ODN诱导NK LU
 ODN1     序列(5′-3′) 实验1 实验2 实验3
细胞自己  0.00  1.26  0.46
 1840  TCCATGTCGTTCCTGTCGTT  2.33  ND  ND
 1960  TCCTGTCGTTCCTGTCGTT  ND  O.48  8.99
 1961  TCCATGTCGTTTTTGTCGTT  4.03  1.23  5.08
 1962  TCCTGTCGTTCCTTGTCGTT  ND  1.60  5.74
 1963  TCCTTGTCGTTCCTGTCGTT  3.42  ND  ND
 1965  TCCTGTCGTTTTTTGTCGTT  0.46  0.42  3.48
 1966  TCGTCGCTGTCTCCGCTTCTT  2.62  ND  ND
 1967  TCGTCGCTGTCTGCCCTTCTT  5.82  1.64  8.32
 1968  TCGTCGCTGTTGTCGTTTCTT  3.77  5.26  6.12
 19792  TCCATGTZGTTCCTGTZGTT  1.32  ND  ND
 1982  TCCAGGACTTCTCTCAGGTT  0.05  ND  0.98
 1990  TCCATGCGTGCGTGCGTTTT  2.10  ND  ND
 1991  TCCATGCGTTGCGTTGCGTT  0.89  ND  ND
 2002  TCCACGACGTTTTCGACGTT  4.02  1.31  9.79
 2005  TCGTCGTTGTCGTTGTCGTT  ND  4.22  12.75
 2006  TCGTCGTTTTGTCGTTTTGTCGTT  ND  6.17  12.82
 2007  TCGTCGTTGTCGTTTTGTCGTT  ND  2.68  9.66
 2008  GCGTGCGTTGTCGTTGTCGTT  ND  1.37  8.15
 2010  GCGGCGGGCGGCGCGCGCCC  ND  0.01  0.05
 2012  TGTCGTTTGTCGTTTGTCGTT  ND  2.02  11.61
 2013  TGTCGTTGTCGTTGTCGTTGTCGTT  ND  0.56  5.22
 2014  TGTCGTTGTCGTTGTCGTT  ND  5.74  10.89
 2015  TCGTCGTCGTCGTT  ND  4.53  10.13
 2016  TGTCGTTGTCGTT  ND  6.54  8.06
1PBMC基本如本文所述。结果是6个分别的实验的代表；每个实验代表不同的供体。2这是甲基化形式的ODN1840；Z=5-甲基胞嘧啶，LU是溶胞单位，ND=未进行，CpG二核苷酸用下划线标出。确定带有对人B细胞繁殖活化为优化的CpG基元的硫代磷酸酯ODN
CpG ODN诱导B细胞繁殖的能力是其附剂潜力的良好指标。具有强附剂效应的ODN一般都诱导B细胞繁殖。为了确定诱导B细胞繁殖的优化的CpG ODN是否与诱导NK细胞活性的ODN相同，测试了相似的一组ODN(表12)。表现出最为一致的刺激性的ODN是ODN2006(表12)。
表12．硫代磷酸酯CpG ODN对人B细胞繁殖的诱导
 DN       序列(5′-3′)               刺激指标1
实验1 实验2 实验3 实验4 实验5
1840 TCCATGTCGTTCCTGTCGTT   4  ND  ND  ND  N
1841 TCCATAGCGTTCCTAGCGTT   3  ND  ND  ND  N
1960 TCCTGTCGTTCCTGTCGTT   ND  2.0  2.0  3.6  N
1961 TCCATGTCGTTTTTGTCGTT   2  3.9  1.9  3.7  N
1962 TCCTGTCGTTCCTTGTCGTT   ND  3.8  1.9  3.9  5
1963 TCCTTGTCGTTCCTGTCGTT   3  ND  ND  ND  N
1965 TCCTGTCGTTTTTTGTCGTT   4  3.7  2.4  4.7  6
1967 TCGTCGCTGTCTGCCCTTCTT   ND  4.4  2.0  4.5  5
1968 TCGTCGCTGTTGTCGTTTCTT   ND  4.0  2.0  4.9  8
1982 TCCAGGACTTCTCTCAGGTT   3  1.8  1.3  3.1  3
2002 TCCACGACGTTTTCGACGTT   ND  2.7  1.4  4.4  N
2005 TCGTCGTTGTCGTTGTCGTT   5  3.2  1.2  3.0  7
2006 TCGTCGTTTTGTCGTTTTGTCGTT   4  4.5  2.2  5.8  8
2007 TCGTCGTTGTCGTTTTGTCGTT   3  4.0  4.2  4.1  N
2008 GCGTGCGTTGTCGTTGTCGTT   ND  3.0  2.4  1.6  N
2010 GCGGCGGGCGGCGCGCGCCC   ND  1.6  1.9  3.2  N
2012 TGTCGTTTGTCGTTTGTCGTT   2  2.8   0  3.2  N
2013 TGTCGTTGTCGTTGTCGTTGTCGTT   3  2.3  3.1  2.8  N
2014 TGTCGTTGTCGTTGTCGTT   3  2.5  4.0  3.2  6
2015 TCGTCGTCGTCGTT   5  1.8  2.6  4.5  9
2016 TGTCGTTGTCGTT   ND  1.1  1.7  2.7  7
1细胞=手术中收集的并储存于-70℃的人脾细胞，或从正常供体采集的PBMC细胞并经过Ficoll离心。细胞培养在96孔U底微滴盘逼供内加入或不加入给定剂量的ODN(加入的剂量为6μml)。N=12个实验。细胞培养4-7天，用1μCi的3H胸苷冲击18小时然后收获并闪烁计数。刺激指数=不加入ODN小孔中的cpm与在整个培养过程中用给定ODN刺激的小孔的cpm的比率(一旦建立培养物后就没有再加入进一步的ODN)。ND=未进行。
确定诱导人IL-2分泌的硫代磷酸酯ODN
CpG ODN诱导IL-12分泌的能力是其附剂潜力的良好指标，特别从其诱导Th1免疫应答这种高度依赖IL-12的能力的角度来讲更是如此。因此，检验了某一组硫代磷酸酯ODN诱导人PBMC体外分泌IL-12的能力(表13)。这些实验显示在某些人PBMC细胞中，大多数CpG ODN可以诱导IL-12的分泌(例如，实验1)。然而，其它的供体只应答少数的CpG ODN(例如，实验2)。从大多数对象来的ODN2006则始终诱导IL-2分泌(表13)。
表13．硫代磷酸酯CpG ODN诱导人IL-12分泌
  ODN1      序列(5′-3′)  IL-12  (Pg/ml)
  实验1   实验2
  细胞自己     0     0
    1962  TCCTGTCGTTCCTTGTCGTT     19     0
    1965  TCCTGTCGTTTTTTGTCGTT     36     0
    1967  TCGTCGCTGTCTGCCCTTCTT     41     0
    1968  TCGTCGCTGTTGTCGTTTCTT     24     0
    2005  TCGTCGTTGTCGTTGTCGTT     25     0
    2006  TCGTCGTTTTGTCGTTTTGTCGTT     29     15
    2014  TGTCGTTGTCGTTGTCGTT     28     0
    2015  TCGTCGTCGTCGTT     14     0
    2016  TGTCGTTGTCGTT     3     0
1PBMC采集于正常供体并用Ficoll离心，然后以106细胞/孔培养在96孔微滴盘中，加入或不加入给定的ODN，如果加入ODN，其剂量为6μg/ml。24小时后收获上清液，用ELISA方法检测IL-12的水平。对每个实验建立标准曲线，其代表不同的供体。确定对B细胞和单核细胞/NK细胞为特异性的寡核苷酸
如图6所示，CpG DNA可以直接活化高纯度B细胞和单核细胞。CpG DNA活化这些种类细胞的机制中有很多相似之处。例如，都需要NFkB活化，对此在下面给予解释。
在对CpG DNA的不同免疫效应的进一步研究中，发现有多于一种以上的CpG基元。具体地讲，寡核苷酸1668带有最佳的小鼠B细胞基元，它是对B细胞和天然杀死细胞(NK)的活化的强诱导物，而寡核苷酸1758是对B细胞的弱活化物，但仍然诱导很好的NK应答(表14)。
表14．刺激优化小鼠B细胞和NK活化的不同的CpG基元
  ODN            序列 B细胞活化1 NK活化2
 1668  TCCATGACGTTCCTGATGCT(SEQ ID NO:44) 42,849  2.52
 1758  TCTCCCAGCGTGCGCCAT(SEQ ID NO:55) 1,747  6.66
 无 367  0.00
CpG二核苷酸用下划线标出，寡核苷酸是合成的并有硫代磷酸酯修饰的主链来改善其核酸酶抗性。1按照实施例1所述用200nM的寡脱氧核苷酸培养48小时后的3H胸苷吸收来进行测定。2按照溶胞单位来测定。免疫刺激核酸的目的论基础
脊椎动物DNA是高度甲基化的，并且CpG二核苷酸是不足表达的。然而，刺激性CpG基元是在微生物基因组DNA中常见的，但是在脊椎动物DNA中就很少见。此外，细菌DNA已经被报道可以诱导B细胞繁殖和免疫球蛋白(Ig)的生产，而哺乳动物DNA则不行[Messina,J.P.et al.,J.Immunol.147:1759(1991)]。在实施例3中进一步描述的实验中，用CpG甲基化酶对细菌DNA的甲基化被发现是消除了有丝分裂的，这就证实了CpG状态的差异是细菌DNA引起B细胞刺激的原因。这个数据支持了下面的结论：即存在于细菌DNA中的未甲基化CpG二核苷酸负责了细菌DNA的刺激效应。
就目的论而言，看起来CpG基元造成的淋巴细胞活化代表了免疫防御机制，其可以区分细菌DNA和宿主DNA。宿主DNA一般由于编程性细胞死亡而存在于很多发炎的组织和区域中，但它通常不诱导或很少诱导淋巴细胞活化，因为CpG被抑制和甲基化。然而，含有未甲基化CpG基元的细菌DNA的存在可以准确地在感染的区域引起淋巴细胞活化，这则是有益的。这种新的活化途径提供了T细胞依赖抗原对特异性B细胞活化的快速替代途径。由于CpG途径通过抗原受体协同了B细胞的活化，带有对细菌抗原为特异性抗原受体的B细胞将通过其细胞膜Ig接受一个活化信号以及从细菌DNA来的第二个信号，并从而倾向于有区分地被活化。这个途径与其它的B细胞活化途径的相互作用提供了使用多克隆抗原来诱导抗原特异性应答的生理学机制。
然而，B细胞活化似乎不是完全非特异性的。带有对细菌产物为特异性抗原受体的B细胞可以通过细胞膜Ig接受一个活化信号，以及从细菌DNA来的第二信号，从而更强烈地启动抗原特异性免疫应答。如同其它的免疫防御机制，对细菌DNA的应答可以具有在某些位置的非理想序列。例如，对自身抗原的自身免疫应答将还倾向于由细菌感染来区分性地启动，因为自身抗原还可以由细菌DNA启动来向自身接受性B细胞提供第二活化信号。确实，细菌感染对自身免疫的诱导是临床观察中常见的。例如，自身免疫系统性红斑狼疮，其本身是1)以产生抗-DNA抗体为特征，2)由抑制DNA甲基转移酶的药物所诱导[Comacchia,E.J.et al.,J.Clin.Invest.92:38(1993)]，以及3)与被减少的DNA甲基化有关[Richardson,B.,L.et al.,Arth.Rheum 35:647(1992)]，所以，它可以部分地由对DNA为特异性的B细胞通过由CpG基元提供的刺激信号的活化来启动，以及由细菌DNA同抗原受体的结合来启动。
此外，脓毒病，其特征是由于大量的对免疫系统的非特异性活化而导致高度发病率和死亡率，在死亡的细菌达到了足以直接活化许多淋巴细胞的浓度而释放出细菌DNA和其它的产物时而活化免疫系统。在脓毒病综合症中CpG DNA作用的进一步证据可见于CoWdery,J.等人的文献[Cowdery,J.,et al.,(1996)TheJournal of Immunolgy 156:4570-4575]。
与抗原通过其表面Ig受体来启动B细胞不同的是，CpG-ODN不诱导任何可检测到的Ca2+流，在蛋白质酪氨酸磷酸化中的改变，或IP3的产生。以带有或不带有CpG基元的ODN与FITC共轭进行的流式细胞仪测定按照Zhao,Q等人所述进行[Amisense Research and Development3:53-66(1993)]，并且显示了相等的膜结合，细胞吸收，外流，以及胞内定位。这就建议，也许不存在对CpG ODN为特异性的细胞膜蛋白质。不同于经过细胞膜来起作用，这些数据建议的是含有未甲基化CpG的寡核苷酸需要细胞吸收来发挥活性：与固体Teflon支持物共价连接的ODN是非刺激性的，如同在抗生物素蛋白小珠或抗生物素蛋白涂布的培养皿上固定化的生物素化的ODN一样。与FITC或生物素共轭的CpG ODN保留了全部的有丝分裂性质，表明没有立体性妨碍。
最近的数据表明，转录因子NFkB的介入是CpG效应的直接或间接的介导物。例如，用CpG DNA处理B细胞或单核细胞15分钟后，NFkB结合活性的水平被提高了(图7)。然而，这种提高不是由于那些不含CpG基元的DNA所导致的。此外，还发现的是，NFkB活化的两个不同的抑制物，即PDTC和胶毒素，完全封闭了CpG DNA对淋巴细胞的刺激，由测定B细胞繁殖或单核细胞分泌细胞因子而证实，这就建议两种细胞都需要NFkB活化。
对NFkB的活化可能有几种机制。它们包括通过对各种蛋白质激酶的活化，或通过反应性氧的产生。没有发现在CpG DNA处理B细胞或单核细胞后立即诱导蛋白质激酶活化的任何证据，而且蛋白质激酶A、蛋白质激酶C，以及蛋白质酪氨酸激酶的抑制物都对CpG诱导的活化没有任何影响。然而，CpG DNA在B细胞和单核细胞中都快速诱导反应性氧的生产，用按照Royall,J.A.所述的敏感性荧光染料二氢若丹明123检测所证实[Royall,J.A.,and Ischiropoulos,H.(Archives of Biochemistry andBiophysics 302:348-355(1993))]。然而，对产生这些反应性样的抑制物完全封闭了NFkB的诱导以及随后的CpG DNA对细胞繁殖和细胞因子分泌的诱导。
下一个问题是，CpG DNA如何快速地导致了反应性氧的产生。本发明人的前述研究证实了寡核苷酸和质粒或细菌DNA被细胞内吸到核内体中。这些核内体迅速地在细胞内被酸化。为了确定这个酸化步骤是否在CpG DNA活化反应性氧的机制中是重要的，用核内体酸化的特异性抑制物封闭该酸化步骤，包括氯喹、莫能菌素、和bafilomycin等，它们通过不同的机制工作。图8A显示了流式细胞仪的研究结果，其中采用了小鼠B细胞以及二氢若丹明123染料来确定反应性氧的水平。该图内在A组中仅仅含有染料的样品显示了细胞对染料的阳性背景水平为28.6％。正如所预期的，反应性氧的这个水平在用PMA和离子霉素处理细胞20分钟后被大大地提高到80％，即阳性对照(B组)。用CpG寡核苷酸处理的细胞也显示反应性氧水平的提高，使大于50％的细胞变为阳性(D组)。然而，用除了CpG改变之外序列完全相同的寡核苷酸处理的细胞没有显示这种显著的反应性氧水平的提高(E组)。
在有氯喹存在时，这些结果非常不同(图8B)。氯喹将细胞中反应性氧的背景水平稍微的降低了一些，使在A组中的未处理的细胞只有4.3％为阳性。氯喹完全消除了在用CpG DNA处理的细胞中的反应性氧的诱导(B组)，但是对用PMA和离子霉素处理的细胞中的反应性氧的水平没有减弱(E组)。这就证明，与PMA加上离子霉素不同，用CpG DNA处理B细胞后的反应性氧的产生需要DNA在核内体中经历酸化步骤。这是淋巴细胞活化的全新的机制。氯喹、莫能菌素、以及bafilomycin还显示对CpG DNA活化NFkB的封闭，以及对随后的繁殖和细胞因子分泌的诱导的封闭。CpG DNA的慢性免疫活化以及自身免疫疾病
CpG DNA对B细胞的活化协同于通过B细胞受体的信号。这就提出了这样的可能，即DNA特异性B细胞可以通过将细菌DNA与其抗原受体结合以及同时的CpG介导的信号的刺激来活化。此外，CpG DNA诱导B细胞变为抗编程死亡的，这种机制被认为在防止对自身抗原如DNA的免疫应答中是重要的。确实，对bDNA的暴露可以启动抗-DNA的抗体的产生。假定CpGDNA具有促进自身免疫的这种潜在能力，那么值得注意的是，患有自身免疫疾病系统性红斑狼疮的患者都具有持续的升高水平的循环血浆DNA，其在高甲基化CpG中被富集。这个发现建议了在狼疮疾病发病机理中CpG DNA造成的慢性免疫活化所可能扮演的角色。
治疗狼疮疾病的一类有效药剂是抗疟疾药物，例如，氯喹。虽然这些药物的治疗机制还不清楚，但是已知的是它们抑制核内体内的酸化。CpG DNA对淋巴细胞的活化不是通过与细胞表面受体的结合所介导的，而是需要细胞吸收来实现的，这种细胞吸收通过将吸收性内吞到酸化的氯喹敏感的胞内小室而发生。这就建议一种假说，即CpG DAN对淋巴细胞的活化可能在发生时相关于酸化的核内体，并且甚至可能是依赖于pH的。为了检验这个假说，采用了对DNA酸化为特异性的抑制物来确定B细胞或单核细胞是否可能在酸化被抑制的条件下应答于CpG DNA。
被检测到的对CpG DNA应答的最早的淋巴细胞活化现象是反应性氧(ROS)的产生，其在脾细胞以及B细胞和单核细胞中都是在5分钟内被诱导出来的。核内体内部酸化的抑制物包括氯喹、bafilomycin A，以及莫能菌素，它们具有不同的作用机制，对CpG诱导的ROS产生有封闭作用，但是对由PMA、与CD40或IgM配合等所介导的ROS的产生没有影响。这些研究显示ROS的产生是通过多种途径的淋巴细胞活化中一种所共有的现象。这种ROS的产生一般独立于核内体中的酸化，而酸化是应答CpGDAN的ROS中所需要的。应答CpG的ROS的生产并不被NFkB抑制物如胶霉毒素所抑制，确认其不是NFkB活化的次级现象。
为了确定CpG DAN在核内体中的酸化是否对其所起到的其它免疫刺激效果是必须的，进行了各种实验。LPS和CpG DNA都诱导相似快速的NFkB活化，提高了原癌基因mRNA水平，以及细胞因子分泌。DNA对NFkB的活化依赖于CpG基元，因为其不被用CpG甲基酶处理的bDAN诱导，也不被因为碱基变换而打乱了CpG的ODN所诱导。采用特异性抗体的超迁移实验指出，被活化的NFkB复合物包括p50和p60组分。并非出人意料的是，在LPS或CpG处理的细胞中的NFkB活化伴随有IkBα和IkBβ的降解。然而，核内体酸化的抑制物选择性地封闭了所有的CpG诱导的活化，但没有对任何LPS诱导的胞内活化现象进行了封闭。值得注意的是，已经确定的是很低浓度的氯喹(＜10μM)抑制了CpG介导的淋巴细胞的活化，因为该剂量远远低于治疗疟疾和记性其它的被报道的免疫效应所需要的剂量(例如，100-1000μM)。这些实验支持了在CpG DNA刺激效果的介导中依赖于pH的信号机制的角色。
  表15．核内体酸化或NFkB活化的抑制物时CpG诱导的
        TNF-α和IL-12表达的特异性封闭
    抑制物
活化物 培养基   Bafilomycin(250nM)   氯喹(2.5μg/ml)   莫能菌素(10nM)   NAC50mM TPCK50μM   胶霉毒素0.1μg/ml    双胶霉毒素0.1μg/ml
TNF-α  IL-12  TNF-α  IL-12 TNF-α  IL-12 TNF-α  IL-12 TNF-α TNF-α     TNF-α     TNF-α
培养基  37  147  46  102  27  20  22  73  10  24     17     41
 CpGODN  455  17,114  71  116  28  6  49  777  54  23     31     441
 LPS  901  22,458  1370  4051 1025 12418  491 4796  417  46     178     1120
表15给出了IL-2和TNF-α的检测：小鼠单核细胞系J774(对IL-12为1×105细胞/毫升，对TNF-α为1×106细胞/毫升)，在有给出的浓度的抑制物或没有抑制物的条件下培养2小时，然后用CpG寡核苷酸(ODN)1826(TCCATGACGTTCCTGACGTTSEQ ID NO:10)刺激，剂量为2μM，对LPS为(10μg/ml)，对TNF-α为4小时，对IL-12为24小时，收集上清液。对上清液用ELISA检测IL-12或TNF-α(pg/ml)，操作参见以前的文献[A.K.Krieg,A.-K.Yi,S.Matson,T.J.Waldschmidt,G.A.Bishop,R.Teasdale,G.Koretzky and D.Klinman,Nature374,546(1995)；Yi,A.-K.,D.M.Klinman,T.L.Martin,S.Matson and A.M.Krieg,J.Immunol,157:5394-5402(1996)；Krieg,A.M.,J.Lab.Clin.Med.,128,128-133(1996)]。用不含有CpG基元的ODN培养细胞没有诱导细胞因子的分泌。在IL-6的检测中也见到了相似的CpG应答的特异性抑制，也见于采用原脾细胞或B细胞系CH12.LX和wEHI-231的实验中。2.5μg/ml氯喹相当于＜5μM。NFkB活化的其它抑制物包括PDTC和钙蛋白酶抑制物Ⅰ和Ⅱ，都给出了相似的抑制效果。给出的数据代表在10个不同的实验中所得到的结果。
过量的CpG基元的免疫活化可能对自身免疫疾病系统性红斑狼疮的发病有作用，其伴随着升高水平的循环高甲基化CpGDNA。氯喹和有关的抗疟疾化合物是对系统性红斑狼疮和其它的一些自身免疫疾病的有效的治疗药物，虽然它们的作用机制尚不明了。我们对极低浓度氯喹可以特异性抑制CpG介导的淋巴细胞活化的能力的确定，建议出了一种新的有效的机制。值得注意的是，狼疮的经常复发被认为是微生物感染启动的。在感染的组织中存在的bDNA水平可以是足以诱导局部发炎应答的。与在脓毒综合症和其它的疾病中CpG DNA起者介导物的作用相结合，我们的研究建议了抗疟疾药物作为核内体酸化的抑制物的新的医药用途。
CpG诱导的ROS生产可能是细胞活化的偶然结果，或者是介导这种活化的信号。ROS的清除物N-乙酰基-L-半胱氨酸(NAC)封闭CpG诱导的NFkB活化，细胞因子的产生，以及B细胞的繁殖，这就建议了在这些途径中ROS生产的起因角色。这些数据与前面的支持ROS在活化NFkB中的角色的证据相吻合。WEHI-231B细胞(5×105细胞/毫升)在有或没有氯喹[5μg/ml(＜10μM)]或胶霉毒素(0.2μg/ml)条件下预培养30分钟。然后对细胞等份试样按照上述在RPMI培养基中10分钟，添加或不添加CpG ODN(1826)或非CpN ODN(1911)1μM，或佛波醇豆蔻乙酸酯(PMA)加离子霉素(iono)。然后对细胞用二氢若丹明123染色并用流式细胞仪分析胞内ROS的产生，具体操作按照以前所述的进行[A.K.Krieg,A.-K.Yi,S.Matson,T.J.Waldschmidt,G.A.Bishop,R.Teasdale,G.Koretzky and D.Klinman,Nature374,546(1995)；Yi,A.-K.,D.M.Klinman,T.L.Martin,S.Matson and A.M.Krieg,J.Immunol,157:5394-5402(1996)；Krieg,A.M.,J.Lab.Clin.Med.,128,128-133(1996)]。J774细胞，一种单核细胞系，显示了相似的依赖pH的CpG诱导的ORS应答。与之相对照，CpG DNA没有诱导胞间ROS的产生，也没有任何可检测的嗜中性白细胞ROS。这些氯喹的浓度(以及那些与其它的核内体酸化抑制物一起使用的浓度)防止了内化的CpG DNA的酸化，用荧光偶联ODN如Tonkinson等人所述的进行了证实[Tonkinson et al.,Nucl.Acids Res.22,4268(1994)；A.M.Krieg,In:Delivery Strategies for Antisense OligonucleotideTherapeutics.Editor,S.Akhtar,CRC Press,Inc.,pp.177(1995)]。用高于抑制核内体酸化所需浓度，观察到了非特异性的抑制效果。每个实验都进行至少三次并得到相似结果。
虽然已知NFkB对基因表达是重要的调节物，但其在对CpGDNA的应答中的角色却始终不清楚。为了确定这个NFkB活化对CpG介导的基因表达是必须的，对细胞在有或没有二硫代氨基甲酸吡咯烷(PDTC)这种IkB磷酸化抑制物存在的条件下用CpG DNA活化。这些NFkB活化的抑制物完全封闭了CpG诱导的原癌基因和细胞因子mRNA和蛋白质的表达，显示NFkB在这些现象中作为介导物的基本作用。在这些研究所用的条件中，没有任何一个抑制物减弱了细胞的生存力。J774，一种小鼠单核细胞系，以5μg/ml的水平在有小牛胸腺(CT)、大肠杆菌(EC)、或甲基化大肠杆菌(mEC)DNA(用CpG甲基化酶进行甲基化4)存在的条件下培养，或与0.75μM的CpG寡核苷酸(ODN1826,表15)或非CpG ODN(TCCATGAGCTTCCTGAGTCT；ODN1745)培养1小时，然后，对细胞进行溶胞，制备核酸提取物。含有共同NFkB位点的双链ODN被5′端放射性标记，用作EMSA的探针，具体操作按照以前所述进行[J.D.Dignam,R.M.Lebovitz and R.G.Roeder,Nucliec Acids Res.11,1475(1983)；M.Briskin,M.Damore,R.Law,G.Lee,P.W.Kincade,C.H.Sibley,M.Kuehl and R.Wall,Mol.Cell.Biol.10,422(1990)]。p50/p65异源双体的位置用对p65和p50的特异性抗体以超迁移方法确定[Santa Cruz Biotechnology,Santa Cruz,CA]。用J774细胞建立氯喹对CpG诱导的但不是LPS诱导的NFkB活化的抑制。将细胞在有或没有氯喹(20μg/ml)存在的条件下预培养2小时，然后按照上述用EC DNA、CpG ODN、非CpG ODN或LPS(1μg/ml)刺激1小时。在B细胞系、WEHI-231和原脾细胞中见到了相似的氯喹敏感性CpG诱导的对NFkB的活化。这些实验都进行三次，氯喹的浓度范围是2.5至20μg/ml，得到了相似的结果。
还明白了的是，CpG刺激的mRNA表达需要核内体酸化以及在B细胞和单核细胞中的NFkB活化。J774细胞(2×106细胞/毫升)在有或没有氯喹[2.5μg/ml(＜5μM)]，或N-甲苯磺酰基-L-苯并氨酸氯甲酮(TPCK,50μM)，这是一种丝氨酸/苏氨酸蛋白酶抑制物，其防止IkB蛋白水解并因此而封闭NFkB活化。然后对细胞进行刺激，使用的是大肠杆菌DNA(EC,50μg/ml)、小牛胸腺DNA(CT,50μg/ml)、LPS(10μg/ml)、CpG ODN(1826,1μM)、或对照的非CpG ODN(1911,1μM),进行3小时。wEHI-231B细胞(5×105细胞/毫升)在有或没有胶霉毒素(0.1μM)或双胶霉毒素(0.1μg/ml)存在的条件下培养2小时，然后进行刺激，使用的是CpG ODN(1826)，或对照的非CpG ODN(1911,TCCAGGACTTTCCTCAGGTT)，用量为0.5μM，时间为8小时。在这两种情况下，细胞被收集后，用RNAzol按照厂商的说明书来制备RNA。进行多探针Rnase保护检测，操作按照以前所述的进行[A.-K.Yi,P.Hornbeck,D.E.Lafrenz and A.M.Krieg,J.Immunol.,157,4918-4925(1996)]。将可比数量的RNA装入每条道内，用核糖体μRNA作为加载对照(L32)。对这些实验重复三次，得到相似的结果。
这些结果表明淋巴细胞对CpG DNA的应答是通过新的途径完成的，涉及了依赖pH的胞内ROS的产生。依赖pH的步骤可能是CpG DNA的运转或加工步骤，ROS的产生，或某些其它的现象。ROS被广泛地认为是在多种类型细胞中的信号途径的第二信使，但是在此之前还没有被证明是可以在B细胞中介导刺激信号的。
假设在核内体之中或其附近有一种蛋白质，它特异性识别含有CpG基元的DNA，并导致反应性氧的产生。为了探测细胞的胞质中能够特异性结合CpG DNA的蛋白质，进行了电泳迁移率变动分析(EMSA)，其中使用的是以5′放射性标记的带有或不带有CpG基元的寡核苷酸。发现有一条带似乎代表与具有CpG基元的单链寡核苷酸特异性结合的蛋白质，但其不与缺少CpG基元的寡核苷酸结合，也不与含有被甲基化了的CpG基元的寡核苷酸结合。这个结合活性在加入了过量的含有NFkB结合位点的寡核苷酸时被封闭。这就建议NFkB或相关的蛋白质是与刺激性CpG寡核苷酸结合的蛋白质或蛋白质复合体的组成部分。
在NFkB被强烈活化的时间点上没有发现CREB/ATF蛋白质被活化。因而，这些数据没有提供NFkB蛋白质实际上结合CpG核酸的证据，而是说明了CpG活性在某种方式上需要该蛋白质。可能的是，CREB/ATF或相关的蛋白质与NFkB蛋白质或其它的蛋白质以某种方式相互作用，这样可以解释CREB蛋白质的结合基元与优化的CpG基元的显著的相似性。还可能的是寡核苷酸结合CREB/ATF或相关的蛋白质，并导致NFkB的活化。
另外，很可能的是，CpG核酸可以与TRAF蛋白质中的一种相结合，该蛋白质与CD40的胞质区结合，并在CD40被交联时调节NFkB活性。这样的TRAF蛋白质的例子包括TRAF-2和TRAF-5。制备免疫刺激性核酸的方法
在本发明中，核酸可以使用本领域熟知的任何数目的方法全程合成。例如，可以采用b-氰乙基氨基磷酸酯法[S.L.Beaucageand M.H.Caruthers,(1986)Tet.Let.22:1859]，核苷H-磷酸酯法[Caregg et al.,(1986)Tet.Let.27:4051-4054；Froehler et al.,(1986)Nucl.Acid.Res.14:5399-5407；Caregg et al.,(1986)Tet.Let.27:4055-4058,Gaffney et al.,(1988)Tet.Let.29:2619-2622]。这些化学合成可以用市售的各种自动化寡核苷酸合成仪还完成。另外，也可以用已知的方法从已经存在的核酸序列(例如，基因组DNA或cDNA)中制备出来，例如，那些利用限制性酶，核酸外切酶和核酸内切酶的技术。
为了用于体内，核酸优选具有相当的抗降解性(例如，对核酸外切酶或核酸内切酶的降解有抗性的)核酸。次级结构，例如茎-环结构，可以稳定核酸来抵抗降解。另外，核酸的稳定化可以通过磷酸酯主链修饰来实现。优选的稳定核酸具有至少部分的硫代磷酸酯修饰的主链。硫代磷酸酯可以用自动化技术由氨基磷酸酯或H-磷酸酯来化学合成。芳基的和酰基的磷酸酯可以通过如美国专利第4,469,863号所描述的方法来制备，并且，芳基磷酸三酯(其中的带电的氧基团被美国专利第5,023,243号和欧洲专利第092,574号所述的方法所烷基化)可以用市售的制剂以自动化固相合成法来制备。进行其它的DNA主链修饰和替换的方法已经被描述了[Uhlmann,E.and Peyman,A.(1990)Chem.Rev.90:544；Goodchild,J.(1990)Bioconjugate Chem.1:165]。带有CpG基元的2′-O-甲基核酸还引起免疫活化，环氧修饰的CpG核酸也如此。事实上，还没有发现任何主链的修饰可以完全消除CpG效应，虽然用5-甲基的C取代C可以极大地消弱之。
为了体内给药，核酸可以结合有能与靶细胞[例如，B细胞，单核细胞和天然杀死细胞(NK)]表面更高度亲和的分子和/或提高靶细胞的细胞吸收而形成“核酸释放复合体”的分子。核酸可以是离子性地或共价地与适宜的分子通过本领域所熟知的技术连接起来。可以使用的有各种的偶联和交联制剂，例如，蛋白质A，碳二亚胺，N-琥珀酰基-3-(2-吡啶二硫基)丙酸盐(SPDP)。核酸还可以用熟知的技术包埋在脂质体或病毒体中。免疫刺激性核酸分子的医疗应用
根据免疫刺激性核酸分子的性质，可以将含有至少一个未甲基化CpG二核苷酸的核酸分子给对象体内给药来治疗“免疫系统缺陷”疾病。另外，含有至少一个未甲基化CpG二核苷酸的核酸分子可以同来自免疫系统缺陷疾病的患者的淋巴细胞(例如，B细胞，单核细胞或NK细胞)在体外接触来活化这些淋巴细胞，然后再将这些淋巴细胞送回到对象体内。
正如本文所描述的，作为对含有未甲基化CpG的核酸分子的应答，有更多数量的脾细胞分泌了IL-6、IL-12、IFN-γ、IFN-α、IFN-β、IL-1、IL-3、IL-10、TNF-α、TNF-β、GM-CSF、RANTES、甚至还有其它的物质。在B细胞、CD4+T细胞和单核细胞中发现了IL-6表达的提高。
免疫刺激性核酸分子还可以同疫苗一起提供给对象来促进其免疫系统并从而产生对疫苗的更好的应答。优选的是，将免疫刺激性核酸分子与疫苗一起或稍微提前一点提供给对象。常规的附剂可以同疫苗一起使用，其一般包括抗原，常规的附剂可以通过提高抗原的吸收来进一步改善免疫疫苗的效果。
如果疫苗是DNA疫苗，则至少有两个成分决定其效率。首先，由疫苗编码的抗原决定免疫应答的特异性。其次，如果质粒的主链含有CpG基元，其就作为疫苗的附剂起作用。因此，CpGDNA起着有效的“危险信号”的作用，并引起免疫系统对该区域内的新抗原产生强烈的反应。这种作用方式似乎是CpG DNA对树枝状细胞和其它的“专业”抗原提呈细胞的局部刺激性效应以及对B细胞的共同刺激效应的结果。
免疫刺激性寡核苷酸和含有未甲基化CpG的疫苗直接活化淋巴细胞并共同刺激抗原特异性应答，这是与常规的附剂(例如，铝沉淀物)完全不同的，常规附剂在单独注射时被认为是通过吸附抗原来起作用并从而使其对免疫细胞更为有效。进而言之，常规附剂只对某些抗原有效，仅仅诱导抗体(体液)免疫应答(Th2)，并且在诱导细胞免疫应答(Th1)方面很差。对于许多的致病物，体液应答对防御仅有很小的作用，有时甚至是有害的。
此外，免疫刺激性寡核苷酸可以在化疗或免疫治疗之前、之中或之后给药，来提高肿瘤细胞对以后的化疗或免疫治疗的应答，或通过诱导恢复性细胞因子如GM-CSF来加速骨髓的恢复。CpG核酸还提高天然杀死细胞的溶胞活性和依赖抗体的细胞毒性(ADCC)。对NK活性和ADCC的诱导可以独自或与其它的治疗一起在癌症的免疫治疗中带来有益效果。
所述的免疫刺激性核酸分子的另外的一种应用是对过敏的去敏感化治疗，过敏一般由对无害的过敏原产生IgE抗体所导致的。由未甲基化CpG核酸诱导的细胞因子主要是属于“Th1”类型的，其最主要的特点是细胞免疫应答并与IL-12和IFN-γ有关。其它的主要类型的免疫应答被称为“Th2”免疫应答，其更多地相关于抗体免疫应答并产生IL-4、IL-5和IL-10。一般来讲，所有的过敏疾病都表现为Th2类型免疫应答介导的和Th1免疫应答介导的自身免疫疾病。根据免疫刺激性核酸分子将对象的免疫应答从Th2(其与IgE抗体的产生和过敏有关)转换为Th1应答(其对抵抗过敏反应)的能力，可以单独向对象提供有效剂量的免疫刺激性核酸或与免疫原一起提供来治疗或防止过敏。
含有未甲基化CpG基元的核酸还具有在治疗气喘中的重要的应用。Th2细胞因子，特别是IL-4和IL-5，都是在气喘病人的呼吸道中呈增高量的。这些细胞因子促进了气喘发炎反应的重要的方面，包括IgE同种型之间的变换，嗜伊红粒细胞趋化性和活化，以及掩饰细胞的生长等。Th1细胞因子，特别是IFN-γ和IL-12，可以对Th2克隆的形成和Th2细胞因子的生产进行抑制。
如同在下面的实施例12中所具体描述的那样，含有未甲基化CpG基元的寡核苷酸(即，TCCATGACGTTCCTGACGTT,SEQ ID NO:11)，但不是对照寡核苷酸(TCCATGAGCTTCCTGAGTCT SEQ ID NO:11，)，可以在小鼠气喘模型中防止发炎细胞浸入和嗜伊红粒细胞化。进而言之，对嗜伊红粒细胞性发炎的抑制是相关于对Th2应答的抑制和对Th1应答的诱导的。
在治疗应用中，有效剂量的适宜的免疫刺激性核酸分子自己或是其与释放复合体的制剂可以用任何能够让寡核苷酸被适宜的靶细胞(例如，B细胞和单核细胞)吸收的方式向对象给药。其它的给药的途径包括注射(皮下、静脉内、肠胃外、腹膜内、胸内，等)。注射可以一次给足，也可以连续渗透。
核酸自己或其与释放复合体一起，都可以与药理学可接受的载体一同给药。在本文中，术语“药理学可接受的载体”指的是那些能够同核酸或核酸释放复合体一起给药并使核酸可以发挥其有关功能的物质。这样的载体的例子包括溶液、溶剂、分散介质、延缓制剂、乳液，等等。根据药理学活性物质来选用这样的介质是本领域熟知的。其它的适合用于核酸的常规载体也都在本发明所包括的范围之内。
术语核酸分子的“有效剂量”指的是对实现所需要的生物学效果为必须的或充足的剂量。例如，对含有至少一个未甲基化CpG的核酸用于治疗免疫系统缺陷来讲，该剂量是对消除肿瘤、癌症、或细菌的、病毒的、或真菌感染为必须的剂量。对于用作疫苗附剂来讲，有效剂量是可以促进对象对疫苗的免疫应答的剂量。对于治疗气喘的“有效剂量”则是讲与气喘相关的Th2类型的免疫应答转变为Th1类型的应答所需要的剂量。对于任何具体的应用的有效剂量，可以根据诸多因素来决定，例如，需要治疗的疾病或状态，具体使用的核酸(例如，含有的未甲基化CpG基元的数目或它们在核酸中的位置)，治疗对象的身高和体重，以及疾病的程度等。本领域的技术人员不需要进行非必须的实验就可以确定具体的寡核苷酸的实际用量。
本发明将由下面的实施例中给予更为细致的描述，这些实施例不从任何角度限制本发明。在本申请全文中所提到的文献(包括文献材料，授权的专利，公开的专利申请，和共悬未决的专利申请)都通过在此引述而合并于本文。实施例
实施例1:ODN对B细胞总RNA合成以及细胞循环的效应
从不带有特异性病原体的6-12周龄的DBA/2或BXSB小鼠(饲养在Iowa大学动物实验中心，没有发现明显的品系差异)的脾中获得B细胞，用抗-Thy-1.2消除T细胞，对淋巴细胞M离心(Cedarlane Laboratories,Hornby,Ontario,Canada)(此后称为“B细胞”)。B细胞中含有少于1％的CD4+或CD8+细胞。8×104个B细胞分三份加入96孔微滴盘中，每孔中含有100μl的RPMI，其中含有10％的FBS(在65℃下热灭活30分钟)，50μM的2-巯基乙醇，100U/ml的青霉素，100μg/ml链霉素，以及2mM的L-谷氨酸。在进行37℃下的20小时培养的开始时刻，加入20μM的ODN，给细胞加以1μCi的3H尿苷，4小时后收获细胞。将全脾细胞用20μM的ODN培养48小时后，对分泌Ig的B细胞用ELISA点检测法计数。表1给出了有关的结果，数据代表与未用ODN培养的细胞比较得到的刺激指数。3H胸苷吸收检测显示了相似的结果，但是有从降解的ODN释放的胸苷造成的非特异性抑制[Matson.S and A.M.Krieg(1992)Nonspecificsuppression of 3H-thymidine incorporation by controloligonucleotides.Antisense Research and Development 2:325]。
       实施例2:ODN对B细胞生产IgM的效应
从新杀死的小鼠获得的脾制备单个细胞的悬浮液，用抗-Thyl，抗-CD4，和抗-CD8和补体处理，具体方法见Leibson等人的文献[Leibson et al.,J.Exp.Med.154:1681(1981)]。按照DeFranco等人的程序[DeFranco et al.,J.Exp.Med.155:1523(1982)]从不连续Percoll梯度的63-70％的带中分离休眠的B细胞(T细胞污染＜0.2％)。这些都按照上述用30μM的ODN或20μg/ml的LPS培养48小时。正在分泌IgM的B细胞数在此时为最大，有ELISA点检测法确定[Klinman,D.M.et al.,J.Immunol.144:506(1990)]。在这个检测中，B细胞在抗-Ig涂布的微滴盘上培养6小时。它们所产生的Ig(＞99％的IgM)用磷酸酶-标记的抗-Ig检测[Southern Biotechnology Associated,Birmingham,AL]。每个B细胞产生的抗体用加入BCIP(Sigma Chemical Co.,St.Louis MO)来观察，在有磷酸酶存在时，形成不溶性兰色沉淀。对细胞进行稀释后，产生了20-40点孔的结果，用其来确定样品中分泌抗体的B细胞的总数。所有的检测都进行三次(数据给出在表1)。在有些实验中，培养物上清液被用ELISA来检测IgM，显示了应答CpG-ODN的相似结果。
        实施例3：细菌DNA对B细胞的刺激
对DBA/2B细胞进行培养，不加入DNA，或加入50μg/ml的a)Micrococcus lysodeikticus；b)NZB/N小鼠脾；c)NFS/N小鼠脾基因组DNA，培养48小时后，加入3H胸苷，4小时后收获细胞。对双份的DNA样品用DNASE I在37℃下酶解30分钟，然后加入细胞培养物。用ELISA-点检测法确定了在48小时的时候，大肠杆菌DNA诱导分泌IgM的B细胞数目提高了8.8倍。
对DBA/2的B细胞进行培养，不加入任何物质，或加入50μg/ml的LPS，或20μM的ODN1、1a、4、或4a。培养细胞并在第4、8、24和48小时收集细胞。对BXSB细胞按照实施例1所述进行培养，分别加入5、10、20、40、80μM的ODN 1、1a、4、4a或LPS。在这个实验中，没加入ODN的小孔中为3833cpm。每个实验都进行至少三次，得到了相似的结果。重复的三个小孔的标准偏差小于5％。
  实施例4:ODN对天然杀死细胞(NK)活性的效应
10×106个C57BL/6脾细胞培养在2ml的RPMI中(按照实施例1进行补充)，加入或不加入40μM的CpG或非-CpG ODN，培养48小时。洗过细胞后，用于在短期51Cr释放检测中作为效应细胞，检测中使用了YAC-1和2C11这两种NK敏感靶细胞系[Balias,Z.K.et al.(1993)J.Immunol.150:17]。加入的效应细胞浓度不同，直至在V底微滴盘中达到每0.2ml含有104个51Cr标记的靶细胞，在37℃温度于5％的CO2下培养4小时。然后离心，对上清液的等份试样计数放射活性。特异性溶胞的百分数的计算是，在有效应细胞存在时的51Cr释放减去靶细胞自己培养时的51Cr释放，与用2％乙酸溶胞后的总释放量减去细胞单独培养时的51Cr cpm释放相比。
    实施例5：对CpG硫代磷酸酯ODN的体外研究
对小鼠称重，腹腔内注射0.25ml的无菌PBS或给定剂量的硫代磷酸酯ODN溶于PBS。24小时后，收获脾细胞，洗过之后染色，进行流式细胞仪测定，使用藻红蛋白偶连6B2，来控制B细胞，以及生物素偶连的抗Ly-6A/E或抗-Iad(Pharmingen,SanDiego,CA)或抗-Bla-1[Hardy,R.R.et al.,J.Exp.Med.159:1169(1984)]。每种条件都实验了两只小鼠，每只小鼠单独进行分析。
  实施例6：硫代磷酸酯ODN对B细胞刺激的效价
用硫代磷酸酯ODN培养B细胞，ODN带有对照ODN1a的序列，或CpG ODN1d的序列，或是带有3Dd的序列，20小时后，加入3H尿苷，或44小时后加入3H胸苷，然后收获细胞并确定cpm。
     实施例7：挽救B细胞干编程性死亡
WEHI-231细胞(5×104个/孔)于37℃下培养1小时，存在或不存在LPS、对照ODN1a、CpG ODN1d、3Dd，然后加入抗-IgM(1μ/ml)。细胞继续培养20小时，加入2μCi/孔的3H胸苷4小时。在这个实验中，没有ODN的细胞或有抗-IgM的细胞得到了90.4×103cpm的3H胸苷吸收。表1中给出的磷酸二酯ODN也给予了相似的保护，虽然由于ODN的降解有一些非特异性抑制。每个实验都进行至少3次，得到相似结果。
        实施例8：体内诱导小鼠IL-6
DBA/2雌性小鼠(2月龄)腹膜内注射500g CpG或对照硫代磷酸酯ODN。在注射后的不同时间点对小鼠采血。每个时间点研究2只小鼠。用Elisa测定IL-6,IL-6的浓度用与标准曲线比较来计算，标准曲线用重组的IL-6获得。该检测的敏感度为10pg/ml。 8小时后无法检测。
      实施例9：系统性诱导小鼠IL-6转录
小鼠和细胞系.DBA/2,BALB/c，和C3H/HeJ小鼠，5-10周龄，用作淋巴细胞来源。所有的小鼠都来自于The JacksonLaboratory(Bar Harbor,ME)，并在Iowa大学的动物饲养中心的无特异性病原体条件下培育和饲养。小鼠B细胞系CH12.LX由Bishop博士(Iowa大学)慷慨提供。
制备细胞。对小鼠折颈处死。从小鼠的脾无菌制备单个细胞的悬浮液。使用抗-Thy-1.2和补体制备消除了T细胞的小鼠脾细胞，并对淋巴细胞M(Cedarlane Laboratories,Hornby,Ontairo,Canada)离心，具体如前人所述[Krieg,A.M.et al.,(1989)A rolefor endogenous retroviral sequences in the regulation of lymphocyteactivation.J.Immunol.143:2448]。
ODN和DNA.磷酸二酯寡核苷酸(O-ODN)和主链修饰的硫代磷酸酯ODN(S-ODN)得自于Iowa大学DNA中心机构或Operon Technologies(Alameda,CA)。大肠杆菌DNA(菌系B)和小牛胸腺DNA购自于Sigma公司(St.Louis,MO)。所有的DNA和ODN都被纯化，使用的是酚∶氯仿∶异戊醇(25∶24∶1)和/或乙醇沉淀。在使用前，对大肠杆菌DNA和小牛胸腺DNA煮沸10分钟，冰冷却5分钟，使它们成为单链。对于某些实验，大肠杆菌DNA和小牛胸腺DNA都用DNaseⅠ(2U/μg DNA)酶解，37℃下2小时，在1X SSC中，有5mM MgCl2存在。为了对大肠杆菌DNA中的CpG二核苷酸内的胞嘧啶甲基化，将大肠杆菌DNA用C:G甲基酶(M.SssⅠ；2U/μg的DNA)于NE缓冲液内处理，补充以160μM的S-腺苷甲硫氨酸，37℃下过夜培养。对甲基化的DNA的纯化按照上述进行。甲基化的效率有HpaⅡ酶解，凝胶电泳分析来确定。所有的酶都购自于New EnglandBiolabs(Beverly,MA)。在ODN中的LPS水平低于12.5ng/ml,大肠杆菌DNA和小牛胸腺DNA中含有少于2.5ng的LPS/mg的DNA，由鲎检测确定。
细胞培养物。所有的细胞都在润湿箱中的5％的CO2内以37℃培养，加入RPMI-1640并补充以10％(v/v)的热灭活小牛血清(FCS),1.5mM L-谷氨酸(50μg/ml),CpG或非CpG磷酸二酯ODN(O-ODN)(20μM)，硫代磷酸酯ODN(S-ODN)(0.5μM)，或大肠杆菌DNA(50μg/ml)或小牛胸腺DNA(50μg/ml),37℃下24小时(对IL-6生产)，或5天(对IgM生产)。刺激物浓度的选择按照以前的研究和滴定来决定。在某些情况下，细胞用CpG O-ODN处理，加入不同浓度(1-10μg/ml)的中性的大鼠IgGl抗小鼠IL-6抗体(杂交瘤MP5-20F3)或对照大鼠IgGl单克隆抗大肠杆菌b-半乳糖苷酶(杂交瘤GL113；ATCC,Rockville,MD)(20),5天。在培养结束时，对培养物上清液用ELISA按照下述进行分析。
体内诱导IL-6和IgM.BALB/c小鼠静脉注射PBS，小牛胸腺DNA(200μg/100μl PBS/小鼠)，大肠杆菌DNA(200μg/100μl PBS/小鼠)，或CpG或非CpG S-ODN(200μg/100μl PBS/小鼠)。小鼠(2只/每组)在不同的时间点后眶采血并折颈处死。取出肝，脾，胸腺，骨髓，从这些器官中用RANzol根据厂商的说明(Tel-Test,Friendswood,TX)制备RNA。
ELISA.平底Immun 1盘(Dynatech Laboratories,Inc.,Chantilly,VA)被涂布，使用的是100μl/孔抗-小鼠IL-6单克隆抗体(MP5-20F3)(2μg/ml)，或抗小鼠IgMμ-链特异性(5μg/ml；Sigma,St.Louis,MO)在碳酸酯-碳酸氢酯，pH9.6缓冲液(15nMNa2CO3,35mM NaHCO3),4℃过夜。然后，用TPBS(0.5mMMgCl2O6H2O,2.68mM KCl,1.47mM KH2PO4,0.14M NaCl,6.6mM K2HPO4,0.5％Tween20)洗该盘，用10％FCS在TPBS中室温下封闭该盘2小时，然后再次洗该盘。培养物上清、小鼠血清、重组的小鼠IL-6(Pharmingen,San Diego,CA)或纯化的小鼠IgM(Calbiochem,San Diego,CA)都用10％FCS进行适宜的稀释，加入到小孔中，三份重复，室温下培育6小时。洗该盘，生物素化大鼠抗-小鼠IL-6单克隆抗体(MP5-32C11,Pharmingen,SanDiego,CA)(1μg/ml在10％FCS中)或生物素化的抗-小鼠Ig(Sigma,St.Louis,MO)以100μl/孔的剂量加入到各个小孔中，室温下培育45分钟，然后用TPBS洗。辣根过氧化物酶(HRP)共轭的抗生物素蛋白(Bio-rad Laboratories,Hercules,CA)以1∶4000的比例在10％FCS(100μl/孔)中稀释，加入到小孔中，室温下培育30分钟。洗盘后，加入苯基二胺二盐酸化物(OPD,Sigma,St.Louis MO),0.05M磷酸盐-柠檬酸盐缓冲液，pH5.0,30分钟。加入0.67N的H2SP4终止反应，用微滴盘读数仪(Cambridge Technology,Inc.,Watertown,MA)在490-600nm处读盘。结果在图1和2中给出。
RT-PCR.有义引物、反义引物、以及IL-6的内部寡核苷酸探针都用公开的序列来合成[Montgomery,RA.and M.S.Dallman(1991),Analysis of cytokine gene expression during fetalthymic ontogeny using the polymerase chain reaction(J.Immunol.)147:554]。cDNA合成以及IL-6的PCR都基本按照Montgomery和Dallman所述的方法进行[Montgomery,R.A.and M.S.Dallman(1991),Analysis of cytokine gene expression during fetal thymicontogeny using the polymerase chain reaction(J.Immunol.)147:554]，采用了RT-PCR试剂，该试剂来自Perkin-Elmer公司(Hayward,CA)。扩增30个循环后分析样品，采用了凝胶电泳和非印迹(unblot)分析方法[Stoye,J.P.et al.,(1991)DNAhybridization in dried gels with fragmented probes:animprovement over blotting techniques,Techniques3:123]。简言之，凝胶在变性缓冲液(0.05M NaOH,1.5M NaCl)中于室温下杂化30分钟，然后在复性缓冲液(1.5M NaCl,1M Tris,pH8)中培育30分钟，再用双蒸水洗。干燥凝胶后，在47℃预杂化2小时，杂化缓冲液(5X SSPE,0.1％SDS)含有10μg/ml的变性大麻哈鱼精子DNA。对凝胶用2×106cpm/ml的IL-6[(5′CATTTCCACGATTTCCCA3′)SEQ ID NO:56]的g-[32P]ATP终端标记的内部寡核苷酸在47℃过夜杂交，室温下洗4次(2XSSC,0.2％SDS)，然后放射性自显影。结果在图3中给出。
细胞繁殖检测。DBA/2小鼠脾B细胞(5×104个细胞/100μl/孔)在37℃下用培养基、CpG或非CpG S-ODN(0.5μM)或O-ODN(20μM)处理24小时。最后4小时，加入[3H]胸苷或[3H]尿苷(1μCi/孔)。吸收的[3H]数量用液相闪烁分析仪(PackardInstrument Co.,Downers Grove,IL)测定。
转染和CAT检测。WEHI-231细胞(107细胞)用电穿孔加入20μg对照的或人IL-6启动子-CAT构建物(由阿肯色大学的S.Manolagas慷慨提供)[Pottratz,S.T.et al.,(1994)17B-estradiolinhibits expression of human interleukin-6 promoter-reporterconstructs by a receptor-dependent mechanism.J.Clin.Invest.93:944],250mV和960μF。电穿孔后，对细胞用各种浓度的CpG或非-CpG ODN刺激。转染16小时后，用溶液检测法来测定氯霉素酰基转移酶(CAT)的活性[Seed,B.and J.Y.Sheen(1988)Asingle phase-extraction assay for chloramphenical acetyl transferaseactivity.Gene76:271]。结果在图5中给出。
     实施例10：决定CpG基元对B细胞刺激程度的
                 寡脱氧核苷酸修饰
采用标准程序在Applied Biosystem公司(Applied BiosystemsInc.,Foster City,CA)的型号为380A、380B、或394的DNA合成仪对ODN进行了合成[Beacage and Caruthers(1981)Deoxynucleoside phosphoramidites--A new class of keyintermediates for deoxypolynucleotide syntheses.TetrahedronLetters22,1859-1862]。对磷酸二酯ODN用标准的β-氰乙基氨基磷酸酯化学方法合成。硫代磷酸酯连接用元素硫氧化亚磷酸连接来加入，而不是采用常规的碘氧化法。四种常见的核苷酸氨基磷酸酯购自于Applied Bisystem公司。所有含OND的磷酸二酯和硫代磷酸酯都用浓氨水在55℃处理12小时来去保护。对ODN用凝胶排阻层析提纯并在使用前冷冻干燥二硫代磷酸酯连接用脱氧核苷酸S-(b-苯甲酰基巯基乙基)吡咯烷硫代氨基磷酸硫酯来加入[Wiesler,W.T.et al.,(1993)In Methods in MolecularBiology:Protocols for Oligonucleotides and Analogs-Synthesis andProperties,Agrawal,S.(ed),Humana Press,191-206.]。含有ODN的二硫酯用浓氨水在55℃处理12小时去保护，然后用反向HPLC提纯。
为了合成在所需要的核苷酸间的连接位置含有甲基硫代磷酸酯或甲基磷酸酯以及磷酸二酯的ODN，采用了两种不同的合成途径。这两种途径的主要区别是对二烷基氨基甲基核苷酸磷化氢采用了偶连试剂，而对甲基硫代磷酸酯采用了氧化试剂。为了合成它们的衍生物，对二烷基氨基甲基核苷酸磷化氢延长了其缩化时间，因为偶连动力学比较缓慢[Jager and Engels,(1984)Synthesis of deoxynucleoside methylphophonates via aphosphonamidite approach.Tetrahedron Letters27,1437-1440]。在偶连步骤完成后，对甲基磷酸二酯用硫化试剂处理[5％元素硫，100mM的N,N-二甲基氨基吡咯烷，存在于二硫化碳/吡啶/三乙胺中]，四个连续的450秒处理，每次都产生了甲基硫代磷酸酯。为了产生硫代磷酸酯连接，对甲基磷酸二酯用标准的氧化试剂处理(0.1M碘，在四氢呋喃/2,6-二甲基吡啶/水中)。
对硅胶结合的寡聚物用蒸馏吡啶/浓氨水为1∶1(v/v)在4℃下处理4天。上清液真空干燥后，溶解于水中，在G50/50的Sephadex柱上色谱。
在本文中，O-ODN指的是磷酸二酯ODN；S-ODN指的是完全硫代磷酸酯修饰的ODN；S-O-ODN指的是嵌合性ODN，其中的中央连接是磷酸二酯，但是5′和3′端的两个连接是硫代磷酸酯修饰的；S2-O-ODN指的是嵌合性ODN，其中的中央连接是磷酸二酯，但5′和3′端的两个连接是二硫代磷酸酯修饰的；MP-O-ODN是嵌合性ODN，其中的中央连接是磷酸二酯，但5′和3′端的两个连接是甲基磷酸酯修饰的。所研究的ODN序列(对CpG二核苷酸用下划线标出)包括：
  3D(5″GAGAACGCTGGACCTTAT),(SEQ ID NO.14)；
  3M(5′TCCATGTCGGTCCTGATGCT),(SEQ ID NO.31)；
  5(5′GGCGTTATTCCTGACTCGCC),(SEQ ID NO.57)；
  6(5′CCTACGTTGTATGCGCCCAGCT),(SEQ ID NO.58)。这些序列基本上代表了在这些研究过程上所测验过的百种CpG和非-CpG的ODN。
小鼠。DBA/2或BXSB来自于The Jackson Laboratory(BarHarbor,ME)，饲养在没有特异性病原体的条件下，用5-10周龄的小鼠作淋巴细胞来源，得到类似的结果。
细胞繁殖检测。对于细胞繁殖检测，将小鼠脾细胞(5×104个细胞/100μl/孔)于37℃的5％CO2润湿箱中培养于RPMI-1640中，补充以10％(v/v)热灭活小牛血清(对O-ODN实验加热到65℃，对仅仅使用修饰的ODN的实验加热到56℃),1.5μM L-谷氨酸，50μM的2-巯基乙醇，100U/ml青霉素和100μg/ml链霉素，按照给出的要求培养24小时或48小时。每个小孔中加入1μCi的3H尿苷或胸苷(按给出的要求进行)，继续培养4小时后，收获细胞。对过滤物用闪烁仪计数。三份重复的小孔的标准偏差小于5％。结果在图6-8中给出。
             实施例11:NK活性的诱导
磷酸二酯ODN购自于Operon Technologies公司(OperonTechnologies,Alameda,CA)。硫代磷酸酯ODN购自于Iowa大学的DNA中心机构，或购自于Midland Cerrified ReagentCompany(Midland,TX)。大肠杆菌(菌系B)DNA和小牛胸腺DNA购自于Sigma公司(Sigma,St.Louis,MO)。所有的DNA和ODN都用酚∶氯仿∶异戊醇(25∶24∶1)萃取和/或乙醇沉淀来纯化。在ODN中的LPS水平低于12.5ng/ml，所含有的大肠杆菌DNA和小牛胸腺DNA低于2.5ngLPS/mg DNA，由鲎检测方法确定。
不含有病毒的4-6周龄的DBA/2小鼠、C57BL/6(B6)小鼠，以及先天无胸腺的BALB/C小鼠都根据合同通过VeteransAffairs来自于National Cancer Institute(Bethesda,MD)。C57BL/6SCID小鼠饲养在Iowa大学动物饲养站的SPF封闭饲养设施中。
人外周血单核淋巴细胞(PBMC)按照前述获得[Ballas,Z.K.et al.,(1990)J.Allergy Clin.Immunol.85:453；Ballas,Z.K.and W.Rasmussen(1990)J.Immunol.145:1039；Ballas,Z.K.and W.Rasmussen(1993)J.Immunol.150:17]。人或小鼠的细胞按5×106个细胞/孔的数量放在24孔微滴盘中的小孔内，培养于37℃的5％CO2的润湿箱中[Ballas,Z.K.et al.,(1990)J.Allergy Clin.Immunol.85:453；Ballas,Z.K.and W.Rasmussen(1990)J.Immunol 145:1039；and Ballas,Z.K.and W.Rasmussen(1993)J.Immunol.150:17]，仅加入培养基，或加入CpG或非CpG ODN，剂量按给出的数据办理，或加入大肠杆菌DNA或小牛胸腺DNA(50μg/ml)并在37℃培养24小时。所有的培养物在18小时后收获，将这些细胞作为效应细胞，按照以前的描述用在标准的4小时51Cr-释放检测中来针对K562(人)或YAC-1(小鼠)靶细胞。在计算溶胞单位(LU)时，1LU定义为30％特异性溶胞所需要的细胞数。如同所给出的那样，在开始培养的时候，加入中性的抗IFN-β抗体(Lee Biomolecular,San Diego,CA)，或IL-12(C15.1,C15.6,C17.8，和C17.15；来自于Giorgio Trinchieri博士，The Wistar Institute,Philadelphia,PA)，或加入它们的同型物对照，直至浓度为10μg/ml。在加入抗-IL-12时，同时加入4中单克隆抗体(MAB)(或它们的同型物对照)各10μg。重组的人IL-2的使用浓度为100U/ml。
  实施例12：在小鼠气喘模型中防止发炎细胞的侵入
            和嗜伊红粒细胞化的发生
6-8周龄的C56BL/6小鼠(来自The Jackson Laboratory,BarHarbor,ME)在第0天和第7天腹膜内注射5000血吸虫卵(Schistosoma mansoni eggs)进行免疫。这些血吸虫卵含有抗原[血吸虫卵抗原(SEA)]，其诱导Th2免疫应答(例如，IgE抗体的生产)。IgE抗体的产生是已知的气喘的重要诱因。
然后，对免疫的小鼠用寡核苷酸(30μg在200μl盐水中，注射)处理，这些寡核苷酸中要么含有未甲基化CpG基元(即，TCCATGACGTTCCTGACGTT；SEQ ID NO.10)，或不含这种基元(即，对照物，TCCATGAGCTTCCTGAGTCT；SEQ ID NO.11)。溶解性SEA(10μg在25μl盐水中)在笫14天和第21天鼻内滴入给药。盐水用作对照。
经过呼吸道的攻击后，在不同的时间点处死小鼠。进行全肺灌洗，收集呼吸道和肺泡的发炎细胞。用ELISA测定灌洗液的细胞因子水平。从全肺洗液中分离RNA，进行Northern分析和RT-PCR研究，使用了CsCl梯度。对肺部组织充气并灌注4％仲甲醛来进行组织学检查。
图9显示的是，小鼠开始被腹膜内注射了血吸虫卵，然后吸入血吸虫卵抗原(空心环)，则在肺部有很多的发炎细胞。然而，当小鼠在开始的时候，与血吸虫卵一起接受了含有未甲基化CpG基元的核酸，则在随后吸入血吸虫卵抗原(空心三角形)后，肺部没有发炎细胞的增多。
图10显示的是，测定肺部灌洗液中嗜伊红粒细胞所获得的相似的结果。嗜伊红粒细胞是与气喘非常相关的发炎细胞。
图11显示，当小鼠在开始的时候用对照寡核苷酸与血吸虫卵一起处理，在吸入了SEA后，对随后的嗜伊红粒细胞侵入肺部没有效应。因此，当小鼠在第14天和第21天吸入血吸虫卵后，它们发生了肺部的急性发炎反应。然而，在第0天和第7天开始接触抗原的时候将CpG寡核苷酸与血吸虫卵一起提供给小鼠的话，则几乎彻底消除了在第14天吸入血吸虫卵抗原所造成的嗜伊红粒细胞的增高。
图12显示，很低剂量的寡核苷酸(＜10μg)就可以提供这样的保护。
图13显示，所发生的发炎应答相关于肺部Th2细胞因子IL-4的水平。
图14显示，提供含有未甲基化CpG基元的寡核苷酸可以实际上将肺部的细胞因子应答改变为IL-12的生产，标明Th1类型的免疫应答。
图15显示，提供含有未甲基化CpG基元的寡核苷酸还可以将肺部的细胞因子应答改变为IFN-γ的生产，标明Th1类型的免疫应答。
实施例13:CpG寡核苷酸诱导人PRMC分泌细胞因子
对Ficoll的Hypaque用常规离心从全血中制备人PBMC。细胞(5×105/ml)在96孔微滴盘内，与10％自身血清，CpG或对照寡核苷酸(对磷酸二酯寡核苷酸为24μg/ml，对核酸酶抗性硫代磷酸酯寡核苷酸为6μg/ml)一起培养，在TNF-α时为4小时，在其它的细胞因子时为24小时，然后收获上清并用ELISA检测，使用Quantikine试剂盒，或使用来自R&D Systems(pg/ml)的试剂，或使用来自Biosource的细胞因子ELISA试剂盒(对IL-12检测)。各检测都按照厂商的说明书进行。表6提供了这些实验的数据，以比没有加入寡脱氧核苷酸的小孔高出的细胞因子水平来表达。
本领域的技术人员将能够采用不超过常规的实验来认识或确定那些本发明描述的具体实施方案的众多等同物。这些等同物都是包括在本发明的权利要求书中的。

Claims (41)

1．一种被分离的核酸序列，其含有至少一个未甲基化CpG二核苷酸，其通式如下：
               5′N1X1CGX2N23′
其中至少一个核苷酸将连续的CpGs分开；X1是腺嘌呤，鸟嘌呤，或胸腺嘧啶；X2是胞嘧啶或胸腺嘧啶；N是核苷酸，并且N1+N2是约0-26个碱基，条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体；并且核酸序列的长度为约8-30个碱基。
2．根据权利要求1所述的核酸序列，其中的X1是胸腺嘧啶。
3．根据权利要求1所述的核酸序列，其中所述的X2是胸腺嘧啶。
4．根据权利要求1所述的核酸序列，其是GTCG(T/C)T或是TGACGTT。
5．根据权利要求1所述的核酸序列，其中的所述的序列是TGTCG(T/C)T。
6．根据权利要求1所述的核酸序列，其中所述的序列是TCCATGTCGTTCCTGTCGTT。
7．根据权利要求1所述的核酸序列，其中所述的序列是TCCTGACGTTCCTGACGTT。
8．根据权利要求1所述的核酸序列，其中所述的序列是TCGTCGTTTTGTCGTTTTGTCGTT。
9．一种被分离的核酸序列，其含有至少一个未甲基化CpG二核酸，其通式如下：
            5′NX1X2CGX3X4N3′
其中至少一个核苷酸将连续的CpGs分开；X1X2选自GpT、GpG、GpA、ApT和ApA；X3X4选自TpT或CpT；N是核苷酸，并且N1+N2是约0-26个碱基，条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体；并且核酸序列的长度为约8-30个碱基。
10．根据权利要求9所述的核酸序列，其中所述的分离至少两个连续的CpGs的核苷酸是胸腺嘧啶。
11．根据权利要求9所述的核酸序列，其中所述的X3和X4是胸腺嘧啶。
12．根据权利要求1或9中的任何一个所述的核酸序列，其中至少一个核苷酸具有磷酸主链修饰。
13．根据权利要求12所述的核酸序列，其中所述的磷酸主链修饰是硫代磷酸酯修饰或磷酸二酯修饰。
14．根据权利要求13所述的核酸序列，其中所述的磷酸主链修饰发生在核酸的5′端。
15．根据权利要求14所述的核酸序列，其中所述的磷酸主链修饰发生在核酸的5′端的前两个核苷酸间的连接。
16．根据权利要求13所述的核酸序列，其中所述的磷酸主链修饰发生在核酸的3′端。
17．根据权利要求16所述的核酸序列，其中所述的磷酸主链修饰发生在核酸的3′端的最后5个核苷酸间的连接。
18．一种刺激对象的免疫活化的方法，其中所述的刺激主要是Th1类型的免疫活化，该方法包括向对象提供具有权利要求1或9所述的核酸序列。
19．根据权利要求18所述的方法，其中所述的对象是人。
20．一种刺激对象的细胞因子生产的方法，该方法包括给对象提供权利要求1或9所述的核酸序列。
21．根据权利要求20所述的方法，其中所述的细胞因子选自包括IL-6、IL-12、IFN-γ、TNF-α和GM-CSF的一组。
22．根据权利要求20所述的方法，其中所述的对象是人。
23．根据权利要求20所述的方法，其中所述的核酸序列选自包括下述序列的一组：
TCCATGTCGCTCCTGATGCT,
TCCATAACGTTCCTGATGCT,
TCCATGACGATCCTGATGCT
TCCATGGCGGTCCTGATGCT
TCCATGTCGGTCCTGATGCT
TCCATAACGTCCCTGATGCT
TCCATGTCGTTCCTGATGCT，以及
TCGTCGTTTTGTCGTTTTGTCGTT。
24．一种刺激对象的NK溶胞活性的方法，该方法包括向对象提供具有权利要求1或9的通式的核酸序列。
25．根据权利要求24所述的方法，其中所述的对象是人。
26．根据权利要求24所述的方法，其中所述的核酸序列选自包括下述序列的一组：
TCGTCGTTGTCGTTGTCGTT,
TCCATGACGGTCCTGATGCT,
TCCATGACGATCCTGATGCT,
TCCATGACGCTCCTGATGCT,
TCCATGACGTTCCTGATGCT,
TCCATAACGTTCCTGATGCT,
TCCATCACGTGCCTGATGCT,
GGGGTCAACGTTGAGGGGGG,
TCGTCGTTTTGTCGTTTTGTCGTT,
TCGTCGTTGTCGTTTTGTCGTT,
GCGTGCGTTGTCGTTGTCGTT,
TGTCGTTTGTCGTTTGTCGTT,
TGTCGTTGTCGTTGTCGTT，以及
TCGTCGTCGTCGTT。
27．一种刺激对象的B细胞繁殖的方法，该方法包括向对象提供具有权利要求1或9的通式的核酸序列。
28．根据权利要求27所述的方法，其中所述的对象是人。
29．根据权利要求27所述的方法，其中所述的核酸序列选自包括下述序列的一组：
TCCTGTCGTTCCTTGTCGTT,
TCCTGTCGTTTTTTGTCGTT,
TCGTCGCTGTCTGCCCTTCTT,
TCGTCGCTGTTGTCGTTTCTT,
TCGTCGTTTTGTCGTTTTGTCGTT,
TCGTCGTTGTCGTTTTGTCGTT，以及
TGTCGTTGTCGTTGTCGTT。
30．一种刺激对象的免疫活化的方法，该方法包括向对象提供具有权利要求1或9的通式的核酸序列，其中所述的核酸序列起附剂的作用。
31．根据权利要求30所述的方法，其中所述的对象是哺乳动物。
32．根据权利要求30所述的方法，其中所述的核酸序列选自包括下述序列的一组：
TCCATGACGTTCCTGACGTT,
GTCG(T/C)T，以及
TGTCG(T/C)T。
33．一种治疗患有气喘疾病的对象的方法，该方法包括向该对象提供处于药理学可接受的载体中的具有权利要求1或9的通式的核酸序列。
34．根据权利要求33所述的方法，其中所述的对象是人。
35．根据权利要求33所述的方法，其中所述的核酸序列是TCCATGACGTTCCTGACGTT。
36．一种通过对CpG介导的淋巴细胞活化的抑制来治疗患有自身免疫疾病或其它的与CpG相关的疾病的对象的方法，该方法包括向该对象提供处于药理学可接受的载体中的核内体酸化抑制物。
37．根据权利要求36所述的方法，其中所述的对象是人。
38．根据权利要求36所述的方法，其中所述的抑制物选自包括bafilomycin、氯喹、和莫能菌素的一组。
39．根据权利要求38所述的方法，其中所述的抑制物的提供剂量小于约10μM。
40．根据权利要求36所述的方法，其中的疾病选自包括系统性红斑狼疮、脓毒症、肠炎、牛皮癣、龈炎、关节炎、克罗恩氏病(Crohn′s disease)、格雷夫斯氏病(Grave′s disease)、和气喘的一组。
41．根据权利要求40所述的方法，其中所述的疾病是系统性红斑狼疮。
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